Compound and application thereof in prevention or treatment of radioactive injury

Abstract

The invention provides a compound and application thereof, and particularly relates to a JWA gene agonist and application of the JWA gene agonist to prevention or treatment of radioactive injury. By activating JWA gene expression and enhancing the cell DNA repairing capacity, the compound has good scavenging capacity on high reactive oxygen generated by X-ray radiation, cell apoptosis caused by radiation can be effectively inhibited so as to relieve damage, and generation of free radicals can be reduced so as to relieve radioactive damage.

Description

A compound and its use in preventing or treating radiation damage

[0001] This disclosure claims priority to Chinese patent application number 202210582602.7, filed with the State Intellectual Property Office of China on May 26, 2022, entitled “A compound and its use for preventing or treating radiation damage.” The entire text of the aforementioned prior application is incorporated into this disclosure by reference. Technical Field

[0002] The present disclosure belongs to the field of medical technology. Specifically, the present disclosure relates to a compound and its use. Further, the present disclosure relates to a JWA gene agonist and its use for preventing or treating radiation damage. Background Art

[0003] Ionizing radiation is a common physical environmental stimulus. It includes gamma and X-rays, neutrons, alpha and beta particles, high-speed electrons, high-speed protons, and other ions that can ionize a given biological or non-biological material upon collision. When living cells absorb ionizing radiation energy, it directly interacts with DNA, disrupting the cell's atomic structure and causing chemical and biological changes. Furthermore, through indirect interactions with water and bystander effects, it produces a range of reactive chemicals that damage nucleic acids, proteins, and lipids.

[0004] Currently, only granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) are FDA-approved anti-radiation drugs for the treatment of acute hematopoietic radiation syndrome. However, there are currently no approved radiation protection drugs for the treatment of radiation syndromes in other parts of the body. Small molecule compounds can be administered orally in tablets or powders, generally do not induce immune responses, and have good patient compliance. Therefore, the development of small molecule compounds that can prevent or treat radiation damage has important clinical value.

[0005] JWA, also known as ARL6IP5 (GenBank AF070523, 1998), is a gene discovered and cloned from a retinoic acid-induced bronchial epithelial cell differentiation model.

[0006] Summary of the Invention

[0007] The present disclosure relates to use of a JWA gene agonist or a pharmaceutical composition thereof in preventing or treating radiation damage.

[0008] In some embodiments, the JWA gene agonist is selected from a compound of formula (I) or a pharmaceutically acceptable salt thereof,

[0009] In some embodiments, the compound of formula (I) is selected from one or a combination of R-JAC4 or S-JAC4, and the structural formula is as follows:

[0010] In some embodiments, the compound of formula (I) is selected from R-JAC4, which has the following structural formula:

[0011] In some embodiments, the radiation injury is selected from one or both of radiation-induced intestinal injury and radiation-induced lung injury.

[0012] In some embodiments, the radiation injury is radiation-induced intestinal injury.

[0013] In some embodiments, the radiation-induced intestinal injury is radiation enteritis.

[0014] In some embodiments, the radiation injury is radiation-induced lung injury.

[0015] In some embodiments, the radiation lung injury is radiation pneumonitis.

[0016] In some embodiments, the radiation-induced lung injury is one or both of radiation-induced oxidative stress injury and radiation-induced DNA damage.

[0017] In some embodiments, the radiation injury is selected from X-ray radiation injury.

[0018] In another aspect, the present disclosure provides a method for preventing or treating radiation damage, comprising administering a therapeutically effective amount of a JWA gene agonist or a pharmaceutical composition thereof to a mammal, preferably a human, in need of such treatment.

[0019] In another aspect, the present disclosure provides use of a JWA gene agonist or a pharmaceutical composition thereof in the preparation of a medicament for preventing or treating radiation damage.

[0020] In another aspect, the present disclosure provides a JWA gene agonist or a pharmaceutical composition thereof for preventing or treating radiation damage.

[0021] In some embodiments, the pharmaceutical composition comprises the JWA gene agonist and pharmaceutically acceptable excipients.

[0022] In another aspect, the present disclosure provides a use of a JWA gene agonist or a pharmaceutical composition thereof in the preparation of a medicament for preventing or treating cancer patients, wherein the medicament is administered in combination with radiation therapy. In another aspect, the present disclosure provides a method for preventing or treating cancer in a mammal, comprising administering a JWA gene agonist or a pharmaceutical composition thereof and radiation therapy to a mammal, preferably a human, in need of such treatment.

[0023] In another aspect, the present disclosure provides use of a JWA gene agonist or a pharmaceutical composition thereof in combination with radiotherapy in preventing or treating cancer.

[0024] In another aspect, the present disclosure provides a combination of a JWA gene agonist or a pharmaceutical composition thereof and radiation therapy for preventing or treating cancer.

[0025] In some embodiments, the cancer is selected from lung cancer.

[0026] In some embodiments, the cancer is selected from non-small cell lung cancer.

[0027] In some embodiments, the cancer is selected from lung adenocarcinoma.

[0028] The compounds disclosed herein activate JWA gene expression, enhance the cell DNA repair ability, have good scavenging ability for highly reactive oxygen species generated by X-ray radiation, can effectively inhibit radiation-induced cell apoptosis and thus reduce damage, and can also reduce the generation of free radicals and alleviate the occurrence of radiation damage. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] FIG1 : Graph showing the effect of whole-body irradiation on the survival rate of mice in Example 1.

[0030] FIG2 is a graph showing the effect of abdominal radiation on the survival rate of mice in Example 2.

[0031] FIG3 : The effect of JAC4 in Example 3 on the spleen and thymus indexes of whole-body irradiated mice.

[0032] Figure 4: HE staining of mouse spleen tissue in Example 3.

[0033] FIG5 : HE staining of mouse thymus tissue in Example 3.

[0034] FIG6 : Blood biochemical results of whole-body irradiated mice treated with JAC4 according to Example 3. ...

[0035] FIG7 is a graph showing the effect of JAC4 in Example 4 on the expression level of JWA in mouse small intestinal epithelial cells.

[0036] FIG8 is a graph showing the dose-effect relationship of the protective effect of JAC4 against whole-body radiation damage in Example 5. FIG8 is a graph showing the dose-effect relationship of the protective effect of JAC4 in Example 5 against whole-body radiation damage.

[0037] FIG9 : The results of JAC4 treatment in Example 5 protecting mice from small intestinal epithelial damage induced by whole-body radiation.

[0038] Figure 10: Prophylactic administration of JAC4 in Example 6 alleviates whole-body radiation-induced small intestinal epithelial damage in mice.

[0039] FIG11 : The therapeutic administration of JAC4 in Example 7 alleviates the damage of the small intestinal epithelium in mice induced by whole-body radiation.

[0040] Figure 12: Diagram showing the results of small intestinal epithelial damage in mice caused by abdominal radiation in Example 8.

[0041] FIG13 shows the results of JAC4 in Example 9 alleviating the weight loss of mice induced by abdominal radiation.

[0042] FIG14 is a graph showing the results of JAC4 in Example 10 alleviating small intestinal epithelial damage induced by abdominal radiation.

[0043] Figure 15: Graph showing the effect of JAC4 of Example 11 on the length of the small intestine and colorectum in mice after abdominal irradiation.

[0044] Figure 16: Graph showing the effects of JAC4 of Example 12 on small intestinal barrier damage, inflammation and oxidative stress response, apoptosis and small intestinal pathological structure in wild-type mice and intestinal epithelial JWA knockout mice induced by abdominal irradiation.

[0045] FIG17 shows the result of JAC4 in Example 13 reducing the release of cytochrome C in IEC-6 cells after radiation.

[0046] FIG18 shows the results of JAC4 in Example 13 reducing the accumulation of reactive oxygen species and apoptosis in IEC-6 cells after radiation.

[0047] FIG19 shows the results of JAC4 in Example 13 alleviating DNA double-strand damage in IEC-6 cells after radiation.

[0048] FIG20 is a graph showing the results of JAC4 in Example 13 alleviating the early apoptosis of IEC-6 cells induced by radiation.

[0049] FIG21 is a graph showing the results of JAC4 in Example 14 alleviating apoptosis in IEC-6 cells.

[0050] Figure 22: NMR spectrum of R-JAC4 of Example 15.

[0051] Figure 23: Mass spectrum of R-JAC4 of Example 15.

[0052] FIG24 is a graph showing the plasma FD4 concentrations of mice in the control group and the drug-treated group in Example 18.

[0053] FIG25 : Statistical results of small intestine length of mice in the control group and the drug-treated group in Example 18.

[0054] Figure 26: Intestinal epithelial cell JWA gene knockout mice (JWA IEC-KO ) and wild-type littermates (JWA IEC-WT ) Graph showing plasma FD4 concentration results.

[0055] Figure 27: Intestinal epithelial cell JWA gene knockout mice (JWA IEC-KO ) and wild-type littermates (JWA IEC-WT ) Results of the levels of pro-inflammatory cytokines TNF-α and IL-1β in plasma.

[0056] FIG28 : JAC4 of Example 20 exerts a synergistic effect in X-ray treatment of the SPCA-1 mouse xenograft subcutaneous tumor model.

[0057] FIG29 shows the results of JAC4 in Example 21 inhibiting irradiation-induced inflammatory response in mice.

[0058] FIG30 : JAC4 of Example 21 inhibits irradiation-induced DNA damage in mouse lung tissue.

[0059] Figure 31: Graph showing the effects of JAC4 combined with irradiation on SPCA-1 and BEAS-2B cells in Example 22.

[0060] Figure 32: Graph showing the effects of JAC4 combined with irradiation on DNA damage in SPCA-1 and BEAS-2B cells according to Example 22.

[0061] Figure 33: Graph showing the effects of JAC4 combined with irradiation on apoptosis of SPCA-1 and BEAS-2B cells according to Example 22.

[0062] FIG34 is a graph showing the results of JAC4 in Example 22 increasing the antioxidant capacity of BEAS-2B.

[0063] FIG35 is a diagram showing the results of JAC4 in Example 22 by preventing NF-κB from entering the nucleus.

[0064] Figure 36: JWA gene deletion in Example 22 attenuates the synergistic and attenuating effects of X-ray on SPCA-1 and BEAS-2B cells.

[0065] Figure 37: Graph showing the effects of JWA gene deletion in Example 22 on irradiation-induced DNA damage and apoptosis in SPCA-1 and BEAS-2B cells.

[0066] FIG38 is a graph showing the results of JAC4 in Example 23 alleviating the damage to the integrity of the intestinal epithelial cell monolayer induced by irradiation. DETAILED DESCRIPTION

[0067] The present disclosure will be further described below in conjunction with specific examples, and the advantages and features of the present disclosure will become clearer as the description progresses. Where specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or the conditions recommended by the manufacturer. Where the manufacturer of the reagents or instruments is not specified, they are conventional products that can be purchased commercially. Unless otherwise defined in the present disclosure, scientific and technical terms related to the present disclosure should have the meanings understood by those of ordinary skill in the art.

[0068] The embodiments of the present disclosure are merely exemplary and do not constitute any limitation on the scope of the present disclosure. It should be understood by those skilled in the art that the details and forms of the technical solutions of the present disclosure may be modified or replaced without departing from the spirit and scope of the present disclosure, but such modifications and replacements shall fall within the scope of protection of the present disclosure.

[0069] The 10-week-old C57BL / 6 mice (weighing 25-30 g) used in this study were obtained from Shanghai Slake Co., Ltd., China, and were SPF-grade animals. The use of animals was approved by the Institutional Animal Care and Use Committee of Nanjing Medical University (IACUC-2004044). X-ray irradiation was performed at the Animal Center of Nanjing Medical University using an Rs-2000Pro X-ray irradiator (RAD SOURCE, USA) at a dose rate of 1.25 Gy / min. The abdominal irradiation range was a 3 cm wide area above the iliac joint.

[0070] In the figures of the present disclosure, * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, **** indicates P < 0.0001, ns and NS indicate no statistically significant difference, # indicates P < 0.05, ## indicates P < 0.01, ### indicates P < 0.001, #### indicates P < 0.0001, ! indicates P < 0.05, and ! ! indicates P < 0.01.

[0071] Abbreviations:

[0072] JAC4: compound of formula (I); R-JAC4: R-form of the compound of formula (I); S-JAC4: S-form of the compound of formula (I); ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; TBI: Total body irradiation; ABI: Abdominal irradiation; BSA: Bovine serum albumin; BER: Base-excision repair; Bcl-2: B-cell lymphoma-2; Bax: Bcl-2 associated X gene; Caspase: Cysteinyl aspartate-specific proteinase; CK-MB: Creatine kinase isoenzymes; CAT: Catalase; DAO: D-amino acid Diamine oxidase; DAPI: 4',6-diamidino-2-phenlindole; DCFH-DA: 2',7'-dichlorodihydrofluorescein diacetate; DMSO: Dimethyl sulfoxide; ELISA: Enzyme-linked immune sorbent assay; FITC-dextran: FITC-labeled dextran; GSH-PX: Glutathione peroxidase; GSH: Glutathione; LDH: Lactic dehydrogenase; PAGE: Polyacrylamide gel electrophoresis; PARP1: Poly(ADR-ribose) polymerase-1; PBS: Phosphate buffered solution; ROS: Reactive oxygen species;SDS: Sodium dodecyl sulfate; SOD: Superoxide dismutase; TEMED: N,N,N',N'-tetramethylethylenediamine; TNF: Trinitrophenol; TBS: Tris-buffered saline; XRCC1: X-ray cross-complementing group 1; IEC: Intestinal epithelial cell.

[0073] Example 1 Effect of JAC4 on the survival rate of mice after whole-body X-ray irradiation

[0074] Ten-week-old C57BL / 6 male mice (weighing 24.8±1.6g) were divided into two groups, 11 in each. After acclimation, they were orally administered JAC4 (100mg / kg) or an equal volume of solvent (polyethylene glycol:ethanol:normal saline = 47.5:7.5:50, v / v / v) once daily for 7 consecutive days. After this treatment, the mice were irradiated with whole-body X-rays (6.5Gy) and continued to receive JAC4 or solvent. After 3 days, JAC4 or solvent treatment was stopped. The mice were observed daily and their weights were recorded. Results were observed 30 days after irradiation, as shown in Figure 1.

[0075] The results showed that JAC4 significantly prolonged the survival time of mice (p < 0.05) (Figures 1B-C) and increased the survival rate of mice after radiation (Figure 1D). Observation results 30 days after radiation showed that the average survival time of mice in the solvent control group after radiation was 7.2 ± 3.1 days, while that of the JAC4-treated group was 17.9 ± 8.4 days (Figure 1C). The results also showed that the JAC4-treated mice also lost weight more slowly after radiation (Figure 1E). Furthermore, all mice in the solvent control group died on day 12 after radiation treatment, while 6 mice died and 5 survived in the JAC4-pretreated group.

[0076] Therefore, JAC4 treatment can significantly prolong the average survival time of mice after whole-body irradiation, improve the survival rate of mice after radiation, and the weight loss of mice in the JAC4-treated group is also slower.

[0077] Example 2 Effect of JAC4 on the survival rate of mice after abdominal X-ray irradiation

[0078] Ten-week-old C57BL / 6 male mice (weighing 26.7±1.0g) were divided into two groups of 10 mice each. After acclimation, they were orally administered JAC4 (100mg / kg) or an equal volume of solvent (polyethylene glycol:ethanol:normal saline = 47.5:7.5:50, v / v / v) once daily for 7 consecutive days. Afterwards, the mice were irradiated with abdominal X-rays (12Gy) and continued to receive JAC4 or solvent. Three days later, treatment with JAC4 or solvent was stopped. The mice were observed daily and their weights were recorded. Results were observed 30 days after irradiation, as shown in Figure 2.

[0079] The results showed that JAC4 significantly prolonged the survival time of mice irradiated with abdominal X-rays (p < 0.05) (Figure 2C) and increased the survival rate of mice after radiation (Figure 2B). Observation results 30 days after radiation showed that the average survival time of mice in the vehicle control group was 8.3 ± 4.2 days, while that of the JAC4-treated group was 16.8 ± 9.0 days (Figure 2C). The results also showed that the weight loss of mice in the JAC4-treated group was slower (Figure 2D). All mice in the vehicle control group died by day 15 after radiation treatment, while at this time, 4 mice in the JAC4-pretreated group had died and 6 mice survived.

[0080] Therefore, JAC4 treatment can significantly prolong the average survival time of mice after abdominal irradiation, improve the survival rate of mice after radiation, and the weight loss of mice in the JAC4-treated group is also slower.

[0081] Example 3 Effects of JAC4 on the spleen, thymus index and hematopoietic system of mice after whole-body irradiation

[0082] Ten-week-old C57BL / 6 male mice (weighing 24.8±1.6 g) were divided into four groups, with eight mice in each group. After acclimation, JAC4 (100 mg / kg) or an equal volume of solvent (polyethylene glycol:ethanol:normal saline = 47.5:7.5:50, v / v / v) was orally administered once a day for 7 consecutive days. Then, one group of mice in the JAC4-treated group and the solvent-treated group were selected to receive whole-body X-ray radiation (6 Gy). The mice continued to receive JAC4 or solvent treatment. After 3 days, JAC4 or solvent treatment was stopped. After 4 days, blood samples, small intestinal tissue, thymus, and spleen tissue were collected. The results are shown in Figure 3.

[0083] The results showed that mice experienced a significant and sustained weight loss from days 1 to 3 after X-ray irradiation, with weight stabilizing on day 4. In the JAC4-treated group, weight loss continued significantly from day 1 to 2 after X-ray irradiation, but began to recover from day 3. The JAC4-treated group experienced significantly less weight loss than the control group (Figure 3B).

[0084] The results of mouse thymus index showed that the thymus index of the solvent group was 2.84±0.45 mg / g, the JAC4 group was 2.51±0.43 mg / g, the solvent + radiation group was 1.25±0.46 mg / g, and the JAC4 + radiation group was 1.63±0.24 mg / g. The thymus index of X-ray irradiated mice was significantly lower than that of the solvent control and JAC4 control groups (p<0.05), while the thymus organ index of X-ray irradiated mice treated with JAC4 was significantly improved and higher than that of the solvent control X-ray irradiation group (p<0.05) (Figure 3C).

[0085] The results of the mouse spleen index showed that the values ​​in the solvent alone group were 3.23±0.31 mg / g, the JAC4 alone group were 3.22±0.26 mg / g, the solvent + radiation group were 1.39±0.12 mg / g, and the JAC4 + radiation group were 1.31±0.18 mg / g. The spleen index of mice was significantly reduced after X-ray radiation (p<0.05), but JAC4 did not improve the spleen organ index (Figure 3D).

[0086] HE staining was performed on the spleen tissue and thymus tissue of each group of mice. The HE staining method was as follows:

[0087] (1) Dewaxing treatment of sections: ① Xylene I treatment for 5 min; ② Xylene II treatment for 5 min; ③ Xylene III treatment for 5 min; ④ 100% ethanol treatment for 5 min; ⑤ 100% ethanol II treatment for 5 min; ⑥ 95% ethanol treatment for 5 min; ⑦ 90% ethanol treatment for 5 min; ⑧ 85% ethanol treatment for 5 min; ⑨ 70% ethanol treatment for 5 min; ⑩ 60% ethanol treatment for 5 min.

[0088] (2) HE staining: After rinsing with double-distilled water, stain with hematoxylin for 6 minutes, separate with 0.5% hydrochloric acid alcohol solution for a while, place in double-distilled water to return to blue, observe under a microscope until the chromatin in the cell nucleus can be clearly observed, and then stain with 0.5% eosin for 30 seconds.

[0089] Figure 4 shows HE staining of mouse spleen tissue. The spleens of the solvent control group and the JAC4-only group without X-ray radiation showed intact structure, with a clear demarcation between the red and white pulps, distinct biochemical centers and margins, and no abnormalities. The spleens of mice treated with X-ray radiation decreased in size, with shrunken capsules, unclear demarcation between the red and white pulps, and significantly reduced white and red pulps. No significant differences were observed between the solvent control group and the JAC4 group (Figures 4A and 4B).

[0090] Figure 5 shows HE staining of mouse thymus tissue. In the solvent control group and the JAC4-only treatment group without X-ray radiation, the thymic lobules were clearly structured, with distinct boundaries between the cortex and medulla. After X-ray radiation, tissue cavities developed, and the boundaries between the cortex and medulla became blurred and difficult to distinguish. However, no significant differences were observed between the solvent control group and the JAC4-treated group (Figures 5A and 5B).

[0091] The biochemical results of the mice were tested by collecting peripheral blood obtained by orbital bleeding into ethylenediaminetetraacetic acid K3 test tubes, and then performing blood biochemical analysis on a hematology analyzer.

[0092] The blood biochemical results of mice are shown in Figure 6: ALT (solvent group: 33.83±11.18 U / L, JAC4 group: 47.17±14.81 U / L, solvent + radiation group: 126.17±14.81 U / L; JAC4+radiation group: 31.33±3.14 U / L), AST (solvent group: 158.83±38.60 U / L, JAC4 group: 140.83±38.72 U / L, solvent + radiation group: 303.83±89.06 U / L; JAC4+radiation group: 110.17±20.86 U / L), LDH (solvent group: 554.33±156.63 U / L, JAC4 group: 444.00±94.58 U / L) L, solvent + radiation group: 2037.67±398.94 U / L; JAC4 + radiation group: 888.67±81.48 U / L), CK-MB (solvent group: 626.00±173.22 U / L, JAC4 group: 565.50±261.37 U / L, solvent + radiation group: 759.67±319.62 U / L; JAC4 + radiation group: 437.67±90.38 U / L), SOD (solvent group: 93.50±12.55 U / mL, JAC4 group: 104.50±9.09 U / mL, solvent + radiation group: 117.17±4.67 U / mL; JAC4 + radiation group: 133.00±6.96 U / mL). The above results showed that after whole-body X-ray irradiation, liver injury indicators ALT and AST increased (p < 0.001) (Figure 6A-B), and myocardial injury indicators LDH, CK-MB, and superoxide dismutase SOD also increased (p < 0.001) (Figure 6C-E), and JAC4 treatment could improve these phenomena.

[0093] Example 4 Effect of JAC4 on X-ray Radiation-Induced Small Intestinal Mucosal Epithelial Damage

[0094] Ten-week-old C57BL / 6 male mice (weighing 24.8±1.6 g) were divided into two groups. After acclimatization, they were orally administered JAC4 (100 mg / kg) or an equal volume of solvent (polyethylene glycol:ethanol:normal saline = 47.5:7.5:50, v / v / v) once a day for 7 consecutive days. After that, they received whole-body X-ray radiation (6 Gy). After another 4 days of JAC4 or solvent treatment, small intestinal tissues were harvested and the expression level of JWA in the small intestine was detected by western blotting. β-actin was used as a control. The grayscale value of JWA / β-actin in (A) was measured and statistically analyzed using Adobe Photoshop CC (**p<0.01). The results are shown in Figure 7.

[0095] The protein immunoblotting (Western Blot) method is as follows:

[0096] 1. Prepare protein samples: Calculate the volume of protein required for a 40 μg / μL concentration based on the protein concentration of each sample. Mix with saline to a final volume of 16 μL. Add 4 μL of 5× SDS sample buffer and mix thoroughly. Centrifuge at 1000×g for 10 seconds, then boil at 100°C for 5 minutes. Cool to room temperature and store at -20°C.

[0097] 2.SDS-PAGE preparation:

[0098] (1) Lower layer separation gel: Install the glass plate and gel rack, and ensure that the bottom of the glass plate is sealed to prevent gel leakage. Prepare separation gels with different configuration ratios according to the molecular weight of the protein to be detected. Add the corresponding ratios of double distilled water, separation gel buffer, 30% polyacrylamide solution, 10% SDS, AP and TEMED in a 50mL test tube, mix thoroughly and immediately inject into the glass plate, and seal with anhydrous ethanol. A clear boundary appears between the separation gel layer and the liquid sealing layer, indicating that the coagulation of the separation gel has been completed, which takes about 1 hour.

[0099] (2) Top stacking gel: Discard the ethanol, rinse repeatedly with double-distilled water 2-3 times, and then use absorbent paper to dry any remaining liquid. In a 50 mL test tube, add 2 mL of double-distilled water, 1 mL of stacking gel buffer, 1 mL of 30% polyacrylamide solution, 40 L of 10% SDS, 20 L of 10% AP, and 8 L of TEMED. Mix thoroughly and inject into the space above the gelled separation gel. Be careful not to create bubbles and slowly insert the sample comb. Allow to polymerize for about 30 minutes at room temperature.

[0100] (3) Remove the glass plate from the mold, place it in the electrophoresis tank, add the pre-prepared electrophoresis solution, and slowly pull out the sample comb.

[0101] 3. Protein loading: After the protein sample is dissolved on ice, shake and mix thoroughly. Add a total volume of 20 μL of sample and 5 μL of protein molecular weight marker to each lane.

[0102] 4. Electrophoresis: Fill up the electrophoresis buffer, connect the power cord, and start electrophoresis. Run the stacking gel at 60V for approximately 40 minutes, and the separating gel at 90V for approximately 2-3 hours.

[0103] 5. Gel cutting: Use a gel cutting board to scrape the gel between the two glass plates, remove the upper layer of concentrated gel and soak it in transfer buffer.

[0104] 6. Transfer: Cut an area of ​​PVDF membrane slightly larger than the gel block and mark it. Activate the membrane by immersing it in methanol for 30 seconds, then place it in 1× transfer buffer to keep it moist. Assemble the transfer "sandwich" in the correct order, with the gel on the black (negative) side and the PVDF membrane on the red (positive) side. Avoid creating bubbles during assembly and clamp the "sandwich" tightly to ensure a smooth transfer. Insert the "sandwich" into the electrophoresis tank and fill it with 1× transfer buffer. Connect the electrophoresis tank wires to the corresponding sockets of the electrophoresis instrument, turn on the electrophoresis instrument, adjust the current and time, and begin the transfer. Because the transfer process generates heat, keep the electrophoresis tank in an ice bath throughout. If the target protein has a molecular weight greater than 100 kDa, transfer at a constant current of 200 mA for 3 hours; if the target protein has a molecular weight less than 100 kDa, transfer at a constant current of 220 mA for 1.5 hours.

[0105] 7. Blocking: Place the PVDF membrane in an appropriately sized incubation chamber, protein side up. Add 5% skim milk powder or 5% BSA to the membrane and block at room temperature for 1 hour. Discard the blocking solution and rinse the PVDF membrane in 1× TBST for 5 minutes.

[0106] 8. Primary Antibody Incubation: Cut the PVDF membrane according to the molecular weight of the target protein and add diluted primary antibody according to the antibody instructions (internal reference Tubulin: Beyotime, 1:1000, Cat. No. AT819, mouse anti-antibody; EGFR: CST, 1:1000, Cat. No. 4267S, rabbit anti-antibody) and incubate with the PVDF membrane on a shaker at 4°C overnight.

[0107] 9. Washing the membrane: Remove the PVDF membrane from the primary antibody dilution solution and wash it with 1×TBST on a shaker at room temperature for 10 min × 3 times.

[0108] 10. Secondary antibody incubation: Incubate the PVDF membrane with the secondary antibody diluted according to the antibody instructions at 37°C for 1 hour.

[0109] 11. Washing the membrane: Remove the PVDF membrane from the secondary antibody dilution solution and wash it with 1×TBST on a shaker at room temperature for 15 min x 4 times.

[0110] 12. Chemiluminescence: Evenly apply 200 μL of the chemiluminescent solution prepared according to the kit requirements on each PVDF membrane, develop and photograph the gel using a chemiluminescence gel imager, and save the image.

[0111] 13. Result processing: The grayscale values ​​of the Western blot bands obtained in the experiment were measured using Adobe Photoshop CC software for semi-quantitative analysis, and the corresponding internal reference bands were used for correction.

[0112] As shown in Figure 7, the average gray value of JWA expression after JAC4 treatment was 0.72±0.13, and the average gray value of JWA expression in the solvent control group was 0.40±0.12, indicating that JAC4 treatment can significantly increase the expression of JWA in the mouse small intestinal epithelial tissue.

[0113] Example 5 Dose-effect relationship of JAC4's protective effect against whole-body X-ray radiation damage

[0114] Ten-week-old C57BL / 6 male mice (weighing 24.8±1.6g) were divided into six groups, namely solvent control group, JAC4 (200mg / kg) treatment group, solvent control group + irradiation group, and JAC4 intervention groups (50, 100, 200mg / kg) + irradiation group, with 12 mice in each group. JAC4 or an equal volume of solvent (polyethylene glycol:ethanol:normal saline=47.5:7.5:50, v / v / v) were orally administered once a day for 7 consecutive days. After that, the mice in the irradiation group received whole-body X-ray radiation (6Gy) and continued to receive oral administration for 3 days before stopping JAC4 or solvent administration.

[0115] The mice were weighed before and after whole-body X-ray irradiation, and the weight curves are shown in Figure 8. As shown in Figure 8, the weight of the mice decreased rapidly after X-ray irradiation, then stabilized, and continued to decrease after the sixth day. The weight loss trend of the mice in the JAC4-treated group was always slower than that in the solvent-irradiated group.

[0116] The small intestinal tissues of mice irradiated with whole-body X-rays were stained with HE. The length of the small intestinal villi in each group was measured and the results were statistically analyzed. The specific method was as follows: HE stained images of the small intestinal tissues of three mice were randomly selected from each group for statistics, and six fields of view were randomly selected from each mouse (**p<0.01, ***p<0.001). The results are shown in Figure 9.

[0117] As shown in Figure 9, the results of small intestinal villus length measurements showed that the length of the villi in the vehicle group was 312.28±14.64μm, while that in the 200mg / kg JAC4 treatment group was 306.78±14.64μm. (P>0.05), indicating that JAC4 treatment alone had no significant effect on small intestinal villus length. On day 3 after X-ray radiation, the length of the villi in the vehicle group was 180.91±31.42μm, while that in the three JAC4 treatment groups was 249.42±27.19μm, 244.78±40.74μm, and 212.43±27.99μm, respectively. Compared with the unirradiated group, the small intestinal epithelium of the irradiated mice was significantly damaged, manifested by a decrease in the number of villi, their fragmentation, and their shortening (P<0.05). Compared with the vehicle plus radiation group, JAC4 treatment at all three doses improved intestinal villus damage (p < 0.05). However, the 50 mg / kg group showed a trend of improvement compared to the 100 and 200 mg / kg groups. Seven days after radiation, intestinal villus length was measured in the vehicle plus radiation group (225.93 ± 39.31 μm), while the three JAC4 dose groups measured 235.57 ± 24.34 μm, 254.95 ± 28.91 μm, and 259.17 ± 17.56 μm, respectively. This suggests that at seven days, the intestinal epithelial stem cells of the mice undergo gradual repair after renewal. Compared with the vehicle plus radiation group, the JAC4-irradiated group had longer intestinal villi. The trends across the three dose groups suggest that JAC4 treatment promotes intestinal villus repair at seven days, with a dose-response relationship.

[0118] Example 6 Protective Effects of JAC4 Prophylactic Administration Against X-Ray Radiation Injury

[0119] Ten-week-old C57BL / 6 male mice (weighing 24.8±1.6g) were divided into four groups and orally administered JAC4 (100mg / kg) or an equal volume of solvent (polyethylene glycol:ethanol:normal saline = 47.5:7.5:50, v / v / v) once daily for 7 consecutive days. The mice were then irradiated with whole-body X-rays (6Gy) and the administration of JAC4 or solvent was stopped. Seven days later, small intestinal tissues were collected for HE staining observation, and the length of small intestinal villi in each group was measured and the results were statistically analyzed. The specific method was as follows: HE staining images of small intestinal tissues of 3 mice were randomly selected from each group for statistics, and 6 fields of view were randomly selected from each mouse (***p<0.001). The results are shown in Figure 10.

[0120] As shown in Figure 10, the length of small intestinal villi was 314.74±22.90μm in the unirradiated vehicle group, 306.54±14.00μm in the JAC4-only treatment group, 181.85±14.00μm in the vehicle + radiation group, and 277.64±33.55μm in the JAC4 pretreatment + radiation group. Compared with the vehicle + radiation group, prophylactic administration of JAC4 significantly improved the reduction in small intestinal villi number, fragmentation, and shortening (p<0.05).

[0121] Example 7: Protective Effects of JAC4 Therapeutic Administration Against X-Ray Radiation Damage

[0122] Ten-week-old C57BL / 6 male mice (weighing 24.8±1.6g) were divided into four groups. After whole-body X-ray radiation (6Gy), the mice were orally administered JAC4 (100mg / kg) or an equal volume of solvent (polyethylene glycol:ethanol:normal saline = 47.5:7.5:50, v / v / v) once a day for 7 consecutive days. The small intestinal tissues were collected for HE staining and the length of the small intestinal villi in each group was measured and the results were statistically analyzed. The specific method was as follows: HE staining images of the small intestinal tissues of 3 mice were randomly selected from each group for statistics, and 6 fields of view were randomly selected from each mouse (***p<0.001). The results are shown in Figure 11.

[0123] As shown in Figure 11, the length of small intestinal villi was 310.86±13.14μm in the unirradiated solvent group, 306.71±24.22μm in the JAC4-only treatment group, 173.16±24.22μm in the radiation + solvent group, and 263.11±37.99μm in the radiation + JAC4 treatment group. Compared with the solvent-irradiated group, treatment with JAC4 after X-ray irradiation also improved the reduction in small intestinal villi number, fragmentation, and shortening (p<0.05).

[0124] Example 8 Intestinal epithelial damage in mice induced by different doses of X-ray radiation

[0125] Ten-week-old C57BL / 6 male mice (weighing 24.8±1.6g) were divided into five groups of six mice each and orally administered JAC4 (100mg / kg) or an equal volume of solvent (polyethylene glycol:ethanol:normal saline = 47.5:7.5:50, v / v / v) once daily for seven consecutive days. Afterwards, the mice received varying doses of abdominal X-ray radiation (3 and 6Gy) and continued treatment with either the drug or the solvent. Four days later, small intestinal tissue was collected for HE staining. Intestinal villus length and ileal crypt height were measured and statistically analyzed for each group. Intestinal histopathology was also scored. Three mice per group were randomly selected for HE staining, and six fields of view were randomly selected from each mouse (***p<0.001). The results are shown in Figure 12.

[0126] As shown in Figure 12, the results of the measurement of small intestinal villus length showed that the non-irradiation control group was 278.62±24.15μm, the solvent+3Gy group was 259.68±32.61μm, and the JAC4+3Gy group was 278.19±25.21μm, p>0.05; the solvent+6Gy group was 187.11±31.70μm, and the JAC4+6Gy group was 278.25±23.79μm, p<0.05. Small intestinal histopathological scores showed that the non-irradiation control group had a score of 1.17±0.41, the vehicle+3Gy group had a score of 2.00±0.63, and the JAC4+3Gy group had a score of 1.50±0.55 (p>0.05); the vehicle+6Gy group had a score of 5.33±1.37, and the JAC4+6Gy group had a score of 4.17±0.98 (p<0.05). Ileal crypt height measurements showed that the non-irradiation control group had a score of 69.12±2.81μm, the vehicle+3Gy group had a score of 50.40±3.26μm, and the JAC4+3Gy group had a score of 62.06±3.26μm (p<0.05). The vehicle+6Gy group had a score of 47.86±3.75μm, and the JAC4+6Gy group had a score of 57.32±4.43μm (p<0.05). The above results show that for the solvent group, 3Gy X-ray radiation caused mild damage to the small intestinal epithelium, with no significant changes within 4 days. After 6Gy X-ray radiation, the small intestinal villi shortened, the crypts became shorter, and the histological score increased, while the above damage was improved in the JAC4-treated group.

[0127] Example 9: JAC4 alleviates abdominal radiation-induced weight loss in mice

[0128] Ten-week-old C57BL / 6 male mice were divided into three groups: a solvent control group, a solvent + irradiation group, and a JAC4 + irradiation group, 8 days after irradiation, with 6 mice in each group. JAC4 (100 mg / kg) or an equal volume of solvent (polyethylene glycol:ethanol:normal saline = 47.5:7.5:50, v / v / v) was orally administered once a day. After 7 days, abdominal X-ray irradiation (12 Gy) was performed and continued for 4 days before stopping. The body weight of the mice was recorded daily before and after whole-body X-ray irradiation, and a body weight curve was plotted. The results are shown in Figure 13.

[0129] As shown in Figure 13B, the weight curve of mice showed that the weight of mice decreased rapidly after abdominal X-ray irradiation and began to rise again on the 7th day, while the weight curve of JAC4 treatment was always above that of the solvent + radiation group.

[0130] Example 10 JAC4 alleviates small intestinal epithelial damage induced by abdominal radiation

[0131] Ten-week-old C57BL / 6 male mice (weighing 24.7±0.9g) were divided into three groups: a vehicle control group (n=6), a vehicle + irradiation group (n=12), and a JAC4 + irradiation group (n=12). JAC4 (100 mg / kg) or an equal volume of vehicle (polyethylene glycol:ethanol:normal saline = 47.5:7.5:50, v / v / v) was administered orally once daily for 7 consecutive days. Afterward, the irradiated mice received abdominal X-ray (12 Gy). Mice in the vehicle control group (n=6), vehicle + irradiation group (n=6), and JAC4 + irradiation group (n=6) were sacrificed 6 hours later, and blood samples and small intestinal tissue were collected. The remaining mice in the vehicle + irradiation group (n=6) and JAC4 + irradiation group (n=6) were sacrificed 8 days later, and blood samples and small intestinal tissue were collected. Abdominal irradiation avoided the femur to minimize damage to the hematopoietic system. The small intestine of each group of mice after abdominal irradiation was stained with HE, and the length of small intestinal villi, the score of small intestinal pathological tissue sections and the height of crypts in each group were measured (*p<0.05, ***p<0.001). The results are shown in Figure 14.

[0132] As shown in Figure 14, ionizing radiation can lead to shortened intestinal villi and increased pathological scores, while JAC4 treatment can protect against radiation-induced damage to the pathological structure of the small intestinal mucosa. The length of the small intestinal villi in the non-irradiated control group was 293.54±22.21μm. Six hours after irradiation, the length of the small intestinal villi in the solvent control group was 194.97±39.39μm, while that in the JAC4-treated group was 308.25±25.65μm (p<0.05). On day 8 after irradiation, the length of the small intestinal villi in the solvent control group was 210.57±20.42μm, while that in the JAC4-treated group was 300.38±33.27μm (p<0.05). The small intestinal histopathology score was 1.17±0.41 in the non-irradiated control group. Six hours after irradiation, the small intestinal histopathology score was 6.33±1.51 in the vehicle control group, while it was 3.17±0.41 in the JAC4-treated group (p<0.05). Eight days after irradiation, the small intestinal histopathology score was 5.00±0.63 in the vehicle control group, while it was 2.83±0.41 in the JAC4-pretreated group (p<0.05). The small intestinal crypt height was 70.42±3.52 μm in the non-irradiated control group. Six hours after irradiation, the small intestinal crypt height was 41.17±5.89 μm in the vehicle control group, while it was 49.80±4.06 μm in the JAC4-pretreated group (p>0.05). On day 8 after irradiation, the height of the small intestinal crypts in the vehicle control group was 52.26±4.88μm, while that in the JAC4 pretreatment group was 53.76±3.33μm (p>0.05). Therefore, abdominal X-ray radiation can lead to shortened small intestinal villi and increased pathological scores. JAC4 treatment can protect against radiation-induced damage to the pathological structure of the small intestinal mucosa and may promote its repair.

[0133] Example 11 JAC4 alleviates X-ray-induced acute radiation enteritis

[0134] Ten-week-old C57BL / 6 male mice (weighing 27.6±1.9g) were divided into three groups, with six mice in each group. They were orally administered JAC4 (100 mg / kg) or an equal volume of solvent (polyethylene glycol:ethanol:normal saline = 40:7.5:52.5, v / v / v) once daily for 7 consecutive days. After that, the mice received abdominal X-ray (12Gy) and continued to receive the drug or solvent treatment. Blood samples and small intestine were collected 4 days later.

[0135] The intestinal epithelial mucosal barrier serves as the interface between luminal microbes and the mucosal immune system, maintaining intestinal barrier homeostasis. The integrity of the intestinal mucosal barrier is determined by multiple factors, including tight junction proteins and other proteins in the cell junction complex. JAC4 has a protective effect on the intestinal mucosal barrier. Disruption of the intestinal mucosal barrier can lead to radiation enteritis, resulting in shortening of the small intestine due to inflammatory edema. The results of mouse small intestinal and colorectal length measurements are shown in Figure 15.

[0136] As shown in Figure 15 , the length of the small intestine in the vehicle-only group was 36.42±2.22 cm, the vehicle-irradiated group was 31.42±3.02 cm, and the JAC4-irradiated group was 34.83±1.44 cm. Compared with the vehicle-only group, the length of the small intestine was shortened after X-ray irradiation, while the length of the small intestine in the JAC4-irradiated group was longer than that in the vehicle-irradiated group (p<0.05) (Figure 15B-C). There was no statistically significant difference in colon length between the groups (Figure 15D) (*p<0.05, **p<0.01, ns: p>0.05).

[0137] Example 12 JAC4 protects mice from X-ray-induced intestinal damage

[0138] JWA flox / + The mice were constructed and embryos were cryopreserved and revived by the Institute of Model Animals of Nanjing University. The mice were of A129 background and were purified to C57 / BL6 background by mating with wild-type mice of C57 / BL6 background for more than 6 generations. Villin-cre mice were provided by Shanghai Southern Model Organisms Company and had C57 / BL6 background. JWA gene knockout (JWA ko ) and littermate controls (JWA wt ) mice are from long-term breeding and conservation, JWA flox / flox Mouse by JWA flox / + Mice were mated and bred to obtain JWA gene knockout in intestinal epithelium (JWA IEC-ko ) and control (JWA IEC-wt ) Mouse by JWA flox / flox The mice were obtained by mating with Villin-cre mice.

[0139] Off-target model using JWA IEC-ko JWA mice and littermates IEC-wt Mice (8 weeks old), irradiated mice were divided into 4 groups: (1) JWA IEC-wt Mouse+solvent group (n=5); (2) JWA IEC-wt Mouse+JAC4 group (n=5); (3) JWA IEC-ko Mouse+solvent group (n=6); (4) JWA IEC-koMice + JAC4 group (n = 5). All mice received 10 Gy abdominal irradiation. Seven days before irradiation, mice were gavaged with 10 mg / kg JAC4 or an equal volume of solvent (polyethylene glycol:ethanol:normal saline = 40:7.5:52.5, v / v / v). Dosage or solvent was continued daily until the model was established. A blank control group (wild-type, n = 6) was not irradiated, and the model was terminated four days after irradiation.

[0140] In order to verify whether the protective effect of JAC4 on X-ray-induced intestinal damage in mice depends on the expression of JWA, the present invention uses JWA IEC-ko The targeting effect of JAC4 was verified in mice.

[0141] JWA IEC-ko and JWA IEC-wt Mice were treated with JAC4 (10 mg / kg) or an equal volume of solvent (polyethylene glycol: ethanol: normal saline = 47.5:7.5:50, v / v / v) daily for 7 days before and 4 days after ABI (10 Gy) exposure. Detection of plasma biomarkers, including intestinal barrier (FD4), inflammation (TNF-α, IL-1β), and oxidative stress (CAT, GSH-px) indicators, showed that compared with the blank control group (4758.4±1066.0 ng / mL), the FD4 content in the solvent group (12865.5±5539.5 ng / mL) was significantly increased after irradiation; while the FD4 content was significantly decreased after JAC4 treatment (6719.9±1823.5 ng / mL). This suggests that 10 mg / kg JAC4 can alleviate irradiation-induced intestinal damage in wild-type mice. In JWA IEC-ko In mice, there was no significant difference in FD4 content after JAC4 treatment (9192.8±5573.2 vs.7673.7±1474.2 ng / mL), indicating that JAC4 plays a role in the JWA induced by irradiation. IEC-ko There was no protective effect on intestinal damage in mice (Figure 16B). At the same time, the levels of TNF-α and IL-1β were consistent with the results of FD4. 10mg / kg JAC4 can reduce JWA after irradiation IEC-wt Inflammatory response in mice (TNF-α: 583.2±78.2 vs. 783.5±192.6 ng / mL; IL-1β: 57.76±5.9 vs. 66.7±7.5 ng / mL), but in JWA IEC-koThere was no similar effect in mice (TNF-α: 769.8±112.3 vs. 668.8±112.7 ng / mL; IL-1β: 65.6±3.3 and 66.7±7.5 ng / mL) (Figure 16C-D). As shown in Figures E and F, ABI increased GSH-Px activity (530.8±207.8 vs. 406.2±197.9 U / mL) and decreased CAT activity (89.4±0.4 vs. 68.7±14.2 U / mL); JAC4 could further enhance JWA. IEC-wt Antioxidant enzymes in mice (815.1±203.9:530.8±207.8U / mL and 85.9±8.2:68.7±14.2U / mL) (P<0.05, P<0.05); however, these results were not IEC-ko Histological analysis confirmed that the villus length and crypt height of wild-type mice in the vehicle group were severely shortened on day 4 after irradiation (villus: 166.3±14.4 vs. 240.7±17.4 μm), (crypt: 55.3±3.7 vs. 76.7±10.0 μm), however, in JWA IEC-ko In mice, JAC4 did not improve intestinal tissue damage (villi: 150.6±8.4 vs. 150.0±11.8μm; crypts: 53.0±3.0 vs. 51.0±3.8μm) (Figure 16G-I). Intestinal tissue was isolated and the expression of apoptotic molecules was analyzed by Western blot (Figure 16J). In wild-type mice, X-ray increased the expression of Bax and cleaved PARP1 in the small intestine compared with the control group. In contrast, JAC4-treated mice downregulated the expression of Bax and cleaved PARP1, while in JWA mice, the expression of Bax and cleaved PARP1 was downregulated. IEC-ko No upregulation of the anti-apoptotic molecule Bcl2 was observed in mice.

[0142] The above results indicate that JAC4 protects the mouse intestinal epithelium from X-ray damage by activating JWA; IEC- ko In mice, JAC4 failed to alleviate irradiated intestinal epithelial damage by targeting JWA. Therefore, JAC4 protection against X-ray-induced intestinal injury in mice depends on JWA expression in intestinal epithelial cells.

[0143] Example 13 Effect of JAC4 on IEC-6 cells

[0144] The in vitro irradiation model of small intestinal epithelial IEC-6 cells was used for observation.

[0145] Preparation of JAC4 (1 μM, 10 μM): Weigh 5 mg of JAC4 and add 1527 μL of DMSO to prepare a 10 mM stock solution; then serially dilute to stock solutions with concentrations of 2 mM and 200 μM, respectively.

[0146] Take 10 μL of 2 mM stock solution and add it to 90 μL of complete culture medium and mix thoroughly. Then add 900 μL of complete culture medium to this 100 μL liquid, mix thoroughly, then add 1 mL of complete culture medium and mix thoroughly before adding to a six-well plate to obtain 10 μM JAC4.

[0147] Take 10 μL of 200 μM stock solution and add it to 90 μL of complete culture medium and mix thoroughly. Then add 900 μL of complete culture medium to this 100 μL liquid, mix thoroughly, then add 1 mL of complete culture medium and mix thoroughly before adding to a six-well plate to obtain 1 μM JAC4.

[0148] Control wells: Take 10 μL of DMSO and add it to 90 μL of complete culture medium and mix thoroughly. Then add 900 μL of complete culture medium to this 100 μL liquid, mix thoroughly, then add 1 mL of complete culture medium and mix thoroughly before adding to the six-well plate.

[0149] IEC-6 cells were pretreated with 1 μM JAC4 or DMSO for 24 h and then irradiated (12 Gy). 24 h later, immunofluorescence images of colocalization of cytochrome c and mitochondria in mitochondria were obtained. Fluorescence colocalization analysis was performed using Image J, and the colocalization correlation coefficient of cytochrome c and mitochondria in IEC-6 cells after different treatments was obtained (***p<0.001). The results are shown in Figure 17.

[0150] As shown in Figure 17, the correlation coefficients for the colocalization of mitochondrial and cytochrome C fluorescence were: 0.84±0.05 for the non-irradiated control group, 0.27±0.06 for the solvent + irradiation group, and 0.73±0.02 for the JAC4 + irradiation group. Therefore, after X-ray treatment of IEC-6 cells, the amount of cytochrome C released from mitochondria increased significantly, with a large amount of cytochrome C released outside the mitochondria. This reduced the colocalization coefficient between mitochondria and cytochrome C, and JAC4 prevented the massive release of cytochrome C.

[0151] IEC-6 cells were pretreated with JAC4 (1 μM) or DMSO for 24 h and then irradiated (12 Gy). 24 h later, the ROS produced in the IEC-6 cells was detected by DCFH probe, the apoptosis of IEC-6 cells was detected by Hoechst staining, and the mitochondrial membrane potential of IEC-6 cells was detected by JC-1 staining. The apoptotic cell proportions of IEC-6 cells after different treatments and the red fluorescence / green fluorescence ratios of IEC-6 cells after different treatments were calculated (**p<0.01, ***p<0.001). The results are shown in Figure 18.

[0152] Among them, the method of Hoechst staining to detect cell apoptosis is as follows:

[0153] 1. IEC-6 cells were cultured in six-well plates at a density of 2 × 10 cells per well. 5 cell.

[0154] 2. After cells adhered, they were treated with JAC4 (1 μM) for 24 h.

[0155] 3.X-ray irradiation treatment.

[0156] 4. Hoechst 33258 (Shanghai Biyuntian) staining, 37°C, 20 min.

[0157] 5. Wash cells three times with PBS.

[0158] 6. Take fluorescence images using a confocal fluorescence microscope. Observe six fields of view in each well and record the average.

[0159] The method for detecting cell apoptosis by ROS staining was as follows: ROS detection kit (Beyotime, Shanghai, China) was used to detect the intracellular ROS content. IEC-6 cells were cultured in six-well plates at a density of 2 × 10 5 After cell attachment, treat the cells with JAC4 (1 μM) for 24 hours. After X-ray irradiation, continue incubation for another 24 hours. Aspirate the culture medium, wash twice with PBS, protect from light, and dilute DCFH-DA with serum-free culture medium. Add at least 600 μL of diluted DCFH-DA to each well and incubate in a cell culture incubator for 25 minutes in the dark. Wash each well three times with 1 mL of serum-free cell culture medium and observe and photograph under a fluorescence microscope.

[0160] Figure 18 shows that after X-ray irradiation, IEC-6 cells generate a large amount of ROS accumulation. Mitochondrial membrane potential was measured by JC-1 staining, and the red / green fluorescence ratio was calculated using Image J. The values ​​for the non-irradiated control group were 0.73±0.09, the solvent-irradiated group was 0.32±0.10, and the JAC4-irradiated group was 0.49±0.05. After X-ray irradiation, mitochondrial membrane potential decreased, while JAC4-treated cells increased mitochondrial membrane potential compared to the radiation-only group. A decrease in membrane potential during the early stages of apoptosis indicates that early apoptosis increases after X-ray irradiation and is relatively reduced after JAC4 treatment. Hoechst staining for apoptosis revealed apoptosis rates of 0.02±0.01% in the non-irradiated control group, 0.23±0.07% in the solvent-irradiated group, and 0.10±0.02% in the JAC4-irradiated group. Therefore, X-ray irradiation increases apoptosis, while JAC4-treated cells decrease it compared to the solvent-irradiated group.

[0161] Cell immunofluorescence images of γ-H2AX in different treatment groups after irradiation of IEC-6 cells were recorded, and the average fluorescence intensity of individual cells was measured using Image J (***p<0.001). The results are shown in FIG19 .

[0162] As shown in Figure 19, the mean fluorescence intensity of the DNA double-strand break marker protein γ-H2AX is: 17.17 ± 1.47 in the non-irradiated control group, 121.67 ± 1.47 in the solvent + irradiation group, and 26.00 ± 1.79 in the JAC4 + irradiation group. Therefore, γ-H2AX fluorescence is enhanced after X-ray irradiation, while fluorescence is weakened after JAC4 treatment.

[0163] Annexin V-PI staining was used to detect apoptosis of IEC-6 cells, and the early apoptosis rates of IEC-6 cells after different treatments were statistically analyzed (***p<0.001). The results are shown in Figure 20. The Annexin V-PI staining method is as follows: 2×10 5 IEC-6 cells were seeded in 6-well plates and treated with 1 μM JAC4 or DMSO for 24 hours after attachment. After 24 hours, cells were harvested in PBS and analyzed using the Annexin V Apoptosis Detection Kit (BD FACS Aria III, USA).

[0164] As shown in Figure 20, the early apoptosis rates were: 3.57±1.41% in the non-irradiated control group, 11.63±1.20% in the vehicle + irradiation group, and 5.74±0.80% in the JAC4 + irradiation group. Therefore, X-ray irradiation increased the early apoptosis rate, while JAC4 treatment decreased it.

[0165] Example 14 JAC4 alleviates IEC-6 cell apoptosis

[0166] IEC-6 cells were pretreated with JAC4 (1 μM, 10 μM) or DMSO for 24 h and then treated with X-rays (12 Gy). 24 h later, the cells were collected and total protein was extracted for western blotting (the same method as in Example 4). The expression of JWA, PARP1 spliceosomes, Bcl-2, Bax, and Caspase-3 spliceosomes in IEC-6 cells was detected. The results are shown in FIG21 .

[0167] As shown in Figure 21, XRCC1 expression in small intestinal tissue and IEC-6 cells decreases after radiation treatment, while JAC4 treatment increases its expression, promoting DNA repair. Ionizing radiation increases the levels of pro-apoptotic proteins PARP1, Caspase-3, Caspase-9, and Bax, while significantly decreasing the levels of anti-apoptotic proteins such as Bcl-2. Therefore, JAC4 treatment can protect against radiation-induced apoptosis, as demonstrated by decreased pro-apoptotic proteins and increased anti-apoptotic proteins compared to the control group.

[0168] Example 15 Preparation of R-JAC4

[0169] Step 1: Preparation of chiral intermediate S1

[0170] SM (10.2 g) was added to methanol (204 ml), stirred and heated to reflux. Dehydroabietylamine (9.68 g) was dissolved in methanol (102 ml). The dehydroabietylamine methanol solution was added dropwise at high temperature. After the addition was complete, the mixture was kept warm and stirred for 30 min. The temperature was naturally lowered to 20-30°C. A white solid precipitated. The mixture was cooled to 0-10°C with ice water to crystallize. The mixture was filtered and the filter cake was rinsed with methanol, dried, and dried in a vacuum oven at 50°C to obtain 7.9 g of an off-white solid S1 with S:R=1.3:98.7.

[0171] The detection conditions for the chiral molecule ratio S:R are as follows:

[0172] Phase A: n-hexane; Phase B: 0.1% diethylamine isopropanol; Chromatographic column: CHIRALPAK AY-H (250 mm x 4.6 mm, 5 μm); Column temperature: 35°C, Flow rate: 1.0 ml / min, Injection volume: 5 μL, Wavelength: 210 nm; Test sample concentration: 1 mg / ml, Solvent: ethanol; A:B (60:40, v / v), Isocratic incubation for 30 min.

[0173] The optical rotation of S1 was tested under the following conditions: sample cell temperature 20°C, sample concentration 1.000 g / 100 cm 3, detection wavelength 589nm, specific rotation result is 59.098°.

[0174] Step 2: Preparation of intermediate S2

[0175] S1 was added to toluene (100 ml) and stirred to disperse. 1 mol / L NaOH (40 ml) and purified water (50 ml) were then added and stirred until a white emulsion formed. The mixture was heated to 55-60°C and stirred for 30 min. The layers were separated while hot. Toluene (50 ml) was added to the aqueous phase and stirred. The layers were allowed to separate. The organic phase was discarded. The aqueous phase was cooled with ice water and concentrated hydrochloric acid (5 ml) was added dropwise to adjust the pH to <1. Ethyl acetate (50 ml) was added and stirred. The layers were separated and the aqueous phase was extracted twice with ethyl acetate (50 ml). The organic phases were combined and dried over anhydrous sodium sulfate. The organic phase was concentrated to 5 ml. Concentration was stopped, the temperature was cooled with stirring, and 30 ml of n-heptane was added dropwise. Solid precipitated. The temperature was then cooled to 0-10°C with ice water to crystallize. The filter cake was filtered with suction, rinsed with n-heptane, and dried to obtain 2.63 g of a light yellow solid with a two-step yield of 25.8%. S:R = 1.07:98.9.

[0176] Step 3: Preparation of intermediate S3

[0177] Methanol (12.5 ml) was added to the reaction flask, and S2 (2.5 g) was added and stirred to dissolve. SOCl2 (1.82 g) was added dropwise at 18°C, and the temperature was maintained at 20-25°C. The addition was completed and the reaction was maintained for 2 h. The raw material reaction was complete. Methanol was concentrated at 30-35°C. After concentration was completed, dichloromethane (12.5 ml) was added, followed by saturated NaHCO3 aqueous solution (7.5 ml). The pH of the aqueous phase was adjusted to 8, and the layers were allowed to stand. The organic phase was washed with 10% NaCl aqueous solution (7.5 ml). After separation, the organic phase was dried over anhydrous sodium sulfate and concentrated to dryness. Ethyl acetate was added and the mixture was concentrated under reduced pressure to 5 mL. The mixture was stirred and cooled. Solids precipitated. N-heptane (30 ml) was added and a large amount of solids precipitated. Ice water was cooled to 0-10°C for crystallization, filtered, and the filter cake was rinsed with n-heptane and dried to give 2.09 g of a white solid with a yield of 77.6%. S:R=0.25:99.75.

[0178] Step 4: Preparation of intermediate S4

[0179] S3 (2.00 g), hydrazine hydrate (1.21 g) and ethanol (40 ml) were mixed, stirred and heated to 25-30 ° C for 3 h. No solid was precipitated. The sample was sent for testing and no raw material remained. The mixture was evaporated at 35-40 ° C to 6 ml, stirred and cooled to 0-10 ° C for crystallization for 1 h. The filter cake was filtered and rinsed with a small amount of ice ethanol, dried and vacuum dried to obtain 1.51 g of white solid with a yield of 75.5%, S:R = 0.25:99.75.

[0180] Step 5: Preparation of intermediate S5

[0181] S4 (1.50 g), N,N'-thiocarbonyldiimidazole (TCDI) (1.65 g) and tetrahydrofuran (15 ml) were added to a three-necked flask, stirred and heated to 55-60 ° C for reaction for 5 h, and samples were taken for inspection. The raw materials were reacted completely, and the reaction solution was concentrated to dryness at 40-45 ° C, and then ethyl acetate (7.5 ml) was added, and the temperature was lowered to 0-10 ° C for crystallization. The filter cake was rinsed with ethyl acetate, dried, and vacuum dried to obtain 1.16 g of off-white solid with a yield of 49.3%, S:R=0.43:99.56.

[0182] Step 6: Preparation of R-JAC4

[0183] S5 (1.56 g), K2CO3 (2.13 g), acetone (15.6 ml), and 2-chloromethylpyridine hydrochloride (1.01 g) were added to a reaction flask, the reaction solution was heated to 40-45°C and reacted for 1.5-2 h, samples were taken for inspection, the raw material reaction was complete, the temperature was lowered to 20-25°C, ethyl acetate (16 ml) and water (16 ml) were added, stirred, and separated, the organic phase was washed with 15% NaCl solution, separated, 10% activated carbon was added to the organic phase for decolorization, filtered, the filter cake was washed with ethyl acetate, the filtrate was combined, concentrated to a volume of 5 ml, stirred, and n-heptane was added dropwise. Solid precipitated, the temperature was lowered to 0-10°C for crystallization, filtered, the filter cake was added to acetone (5 ml), stirred and dispersed, and purified water (15 ml) was added, cooled to 0-10°C for slurrying, filtered, and the filter cake was vacuum dried to obtain 1.34 g of off-white solid powder with a yield of 79.5%.

[0184] DMSO-D6δ H :8.51(d,J=4.3Hz,1H),7.76(d,J=7.7,1.8Hz,1H),7.47(d,J=7.8Hz,1H),7.36–7.26(m,1H),7.01- 6.95(m,1H),6.94-6.86(m,3H),5.81(dd,J=4.9,2.9Hz,1H),4.64(s,2H),4.59–4.48(m,2H)(Figure 22);

[0185] LC / MS (m / z, MH + ):328.2(Figure 23).

[0186] Example 16 Preparation of S-JAC4

[0187] The structure of S-JAC4 is shown in the following formula (II):

[0188] S-JAC4 was obtained by chiral column separation from the racemate JAC4 under the following conditions:

[0189] Instrument: MGⅡ preparative SFC (SFC-1)

[0190] Column: ChiralPak AS, 250 × 30 mm ID, 10 μm

[0191] Mobile phase: A is CO2, B is isopropanol (0.1% NH3H2O)

[0192] Gradient: B 35%

[0193] Flow rate: 80 mL / min

[0194] Back pressure: 100 bar

[0195] Column temperature: 38°C

[0196] Wavelength: 220nm

[0197] Cycle time: 5 minutes

[0198] Sample preparation: Dissolve the compound in 100 ml of methanol / DCM

[0199] Injection: 2 ml per injection.

[0200] Post-treatment: The separated fractions were dried by rotary evaporation at a bath temperature of 40°C to obtain the desired isomer.

[0201] Among them, the synthetic route of JAC4 racemate is as follows:

[0202] Example 17 Pharmacokinetic Properties of R-JAC4 and S-JAC4

[0203] Experimental animals:

[0204] Healthy adult BALB / c female mice were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd. (Animal Production License No.: SCXK(Beijing)2016-0006). They were randomly divided into two groups of three mice each and administered intravenously and orally.

[0205] Drug preparation:

[0206] Compounds R-JAC4 and S-JAC4 were weighed and gradually dissolved in a volume ratio of 5% DMSO, 40% triglyceride (TG), and 55% aqueous solution containing 7.5% (w / v) sulfobutyl-β-cyclodextrin sodium salt to a concentration of 0.5 mg / mL for intravenous injection;

[0207] According to the same method as above, the concentration was prepared to 1 mg / mL for oral administration.

[0208] Dosage:

[0209] Intravenous group: BALB / c mice were fasted overnight and administered by gavage. The dose was 3 mg / kg and the administration volume was 6 mL / kg.

[0210] Oral gavage group: BALB / c mice were fasted overnight and administered via tail vein. The dose was 10 mg / kg and the administration volume was 10 mL / kg.

[0211] How to do it:

[0212] After oral or intravenous administration, 40 μL of blood was collected from the mice's orbits at 5 min, 15 min, 30 min, 1 h, 2 h, 6 h, and 24 h, and added to EDTA-2K anticoagulant tubes. The blood was centrifuged at 12,000 rpm for 5 min in a 4°C centrifuge to separate the plasma, and the separated plasma was stored at -20°C.

[0213] The levels of the test compound in mouse plasma were determined after intravenous or oral administration of different drug doses. The following steps were performed: thaw the plasma sample at room temperature and vortex for 1 minute. Quantitatively transfer 10 μL of the sample to a 2 mL 96-well plate. Add 50 μL of internal standard solution (tolbutamide in acetonitrile) and 50 μL of precipitant (acetonitrile:methanol = 7:3 (v / v)). The plate was shaken at 1000 rpm for 3 minutes and centrifuged at 4000 rpm for 15 minutes. Then, transfer 20 μL of the supernatant to a 2 mL 96-well plate, dilute with 280 μL of water, shake at 1000 rpm for 3 minutes, and sample analysis was performed. At the end of the model, the mice were euthanized.

[0214] LC-MS / MS conditions: Column: Waters T3 1.8 μm (2.1 mm x 30 mm); Mobile phase A: 0.1% formic acid in water, Mobile phase B: 0.1% formic acid in acetonitrile; Column temperature: 40°C; Injection volume: 5 μL; Flow rate: 0.6 mL / min; Gradient elution program: see Table 1.

[0215] Table 1 Gradient elution program

[0216] The pharmacokinetic properties of the test compounds are shown in Tables 2 and 3.

[0217] Table 2 Pharmacokinetic parameters after single intravenous administration of the compound (Mean ± SD, n = 3)

[0218] Table 3 Pharmacokinetic parameters after single oral administration of the compound (Mean ± SD, n = 3)

[0219] According to the bioavailability formula F = (AUC-po / Dose-po) / (AUC-iv / Dose-iv), where AUC-po represents the area under the oral dose curve, AUC-iv is the area under the intravenous injection curve, Dose-po is the oral dose, and Dose-iv is the intravenous dose, calculation shows that the oral bioavailability of R-JAC4 is higher than that of S-JAC4.

[0220] Example 18 Pharmacological and pharmacodynamic effects of R-JAC4 in a mouse model of radiation enteritis

[0221] Ten-week-old male C57 / BL6 mice were selected and randomly divided into four groups before the experiment: a normal control group (i.e., mice not subjected to any drug administration or irradiation treatment), a solvent control group, an R-JAC4 10 mg / kg dose group, and an R-JAC4 3 mg / kg dose group. The R-JAC4 solvent formula was polyethylene glycol:ethanol:normal saline = 40:7.5:52.5, v / v / v. The solvent group and the drug administration group were administered the same volume of solvent via the same route.

[0222] The solvent control group was given the solvent by gavage, and the R-JAC4 treatment group was given R-JAC4 10 mg / kg and R-JAC4 3 mg / kg by gavage. The drugs were administered prophylactically 7 days before X-ray irradiation and were given once a day until the day before the end of the experiment, for a total of 7 doses.

[0223] X-ray irradiation: Mice were anesthetized with peritoneal X-rays and irradiated with 12 Gy (1.25 Gy / min) to the entire abdomen. The irradiation area was 3 cm below the chest and above the iliac joints to induce gastrointestinal radiation syndrome.

[0224] Small intestinal permeability test: On the 4th day after radiation, mice were gavaged with FITC-dextran 4kDa, abbreviated as FD4, at a dose of 0.6 mg / g body weight. Four hours later, peripheral blood plasma was collected in anticoagulant tubes and the plasma was obtained by centrifugation. 100 μl of sample was added to each well of a 96-well plate, and the fluorescence intensity was measured (excitation 485 nm, emission wavelength 535 nm). The FD4 concentration of each sample was calculated based on the regression of the standard curve. The experimental results are shown in Figure 24.

[0225] The results showed that the penetration of the fluorescent molecule FD4 in the normal solvent control group was higher than that in the drug-treated group. The compound R-JAC4 10 mg / kg and 3 mg / kg showed a dose-dependent downregulation of the concentration of fluorescent FD4 in the plasma of radiation enteritis model mice, indicating that R-JAC4 administration can reduce intestinal epithelial damage in mice and protect the intestinal epithelial barrier function.

[0226] Small intestine length measurement: At the end of the experiment, the mice were euthanized and dissected, and the intact small intestine was taken out and placed on graph paper to measure the length of the small intestine. The experimental results are shown in Figure 25.

[0227] The results showed that compound R-JAC4 could effectively reduce the degree of small intestinal shortening in a dose-dependent manner.

[0228] Example 19 Study on the Anti-Inflammation and Anti-Oxidation Effects of R-JAC4

[0229] Intestinal epithelial cell JWA gene knockout mice (JWA IEC KO ) and wild-type littermates (JWA IEC WT A radiation enteritis model was established in the same manner as in Example 17, and R-JAC4 10 mg / kg was administered. At the experimental endpoint, in addition to measuring FD4 concentration, plasma was collected and assayed for inflammatory cytokine levels using ELISA, and biochemical assays were performed to measure antioxidant enzyme activities. The results are shown in Figures 26 and 27.

[0230] The results show that: IEC WT In the mouse radiation enteritis model, R-JAC4 10mg / kg administration significantly downregulated the plasma FD4 level, while in JWA IEC KO There was no significant change in FD4 levels in mice compared with the corresponding solvent control group; JWA IEC WT In the mouse radiation enteritis model, R-JAC4 10mg / kg administration significantly downregulated the levels of plasma inflammatory cytokines TNF-α and IL-1β, whereas in JWA IEC KO There was no significant change in the R-JAC4-treated group compared with the corresponding solvent control group on mice; JWA IEC WTIn the mouse radiation enteritis model, R-JAC4 10mg / kg administration significantly upregulated the activities of plasma glutathione peroxidase and catalase, whereas in JWA IEC KO There were no significant changes in the R-JAC4-treated mice compared to the corresponding vehicle control group. Therefore, R-JAC4 can exert anti-inflammatory and antioxidant functions through the JWA-related signaling pathway, thereby playing a therapeutic role in mice with radiation enteritis.

[0231] In summary, in the present disclosure, on the one hand, JAC4 can increase the transcription of XRCC1 and inhibit the degradation of XRCC1 after activating JWA, thereby repairing the DNA damage of small intestinal cells damaged by X-rays through the BER signaling pathway; on the other hand, JWA, as an effective response gene, increases the expression of glutathione peroxidase and superoxide dismutase, reduces the production of intracellular malondialdehyde, and thus reduces the content of intracellular reactive oxygen species; protects the mitochondrial membrane, reduces the release of cytochrome C, and at the same time inhibits the pro-apoptotic protein Bax, activates the anti-apoptotic protein Bcl-2, and ultimately inhibits the production of Caspase-9 and Caspase-3 splicing bodies, thereby effectively reducing the occurrence of mitochondrial apoptosis in small intestinal cells and exerting a protective effect on small intestinal epithelial cells after radiation.

[0232] Example 20 JAC4 exerts a synergistic effect on X-ray irradiation therapy in a subcutaneous lung cancer xenograft tumor model

[0233] SPCA-1 cells in the logarithmic growth phase were taken, 5×10 6 100 μL was injected subcutaneously into 4-5 week old male BALB / C nude mice at the same level as the lungs. When the tumor volume reached 60-100 mm 3 (tumor volume = length × width 2 At 24 hours ( / 2), mice were randomly divided into a solvent control group, a 100 mg / kg JAC4 group (daily), a 3 Gy x 5 X-ray irradiation group (5 consecutive days of irradiation), and a 100 mg / kg JAC4 + 3 Gy x 5 X-ray irradiation group, with 6 mice in each group. The JAC4 solvent formulation consisted of polyethylene glycol:ethanol:normal saline (40:7.5:52.5, v / v / v). The solvent and treatment groups were administered the same volume of solvent via the same route. Mice were anesthetized with isoflurane inhalation via a mask and placed in the supine position. The irradiation area was adjusted to the tumor and the entire lung using a width indicator light and an internal beam limiter, with a width of approximately 2 cm. The hatch was closed, and the irradiation time was adjusted according to the dose rate to approximately 126 seconds, resulting in a total dose of 3 Gy. The irradiator was then activated for irradiation. JAC4 (100 mg / kg) was administered orally daily. Body weight and tumor volume were measured daily. The model was terminated 13 days after dosing.

[0234] After the model was completed, the tumor tissues were stained with HE. The specific method was as follows: 3 mice tumor tissues were randomly selected for HE staining in each group, and 6 fields of view were randomly selected for each mouse.

[0235] As shown in Figure 28 , the results of the SPCA-1 cell xenograft mouse model showed that tumor volumes in the 100 mg / kg JAC4 and irradiation treatments were significantly smaller than those in the control group, while the combined treatment group exhibited superior tumor suppression compared to either group (Figures 28C, D). Compared to the X-ray treatment alone, the ratio of tumor weight to body weight in the combined treatment group was significantly reduced (Figure 28E). Compared to the control group, the tumor suppression rates for 100 mg / kg JAC4 treatment, irradiation treatment, and combined treatment were 25.45%, 49.95%, and 78.05%, respectively (Figure 28F). Furthermore, there were no statistically significant differences in body weight among the groups, and no mortality occurred, demonstrating that JAC4 has no significant toxic side effects on mice (Figure 28G). Furthermore, H&E staining of tumor tissue from xenografted animals revealed sparsely arranged cells and numerous interstitial spaces in the JAC4 combined with X-ray treatment group compared to the other treatment groups, demonstrating that the combination of JAC4 and X-ray promotes tumor necrosis, leading to significant tissue necrosis in the central region of the transplanted tumor (Figure 28H). The above results show that JAC4 plays a synergistic role in X-ray treatment of the SPCA-1 mouse xenograft subcutaneous tumor model.

[0236] Example 21 JAC4 inhibits X-ray-induced inflammatory response and lung DNA damage in mice bearing lung cancer xenografts

[0237] After the model described in Example 20 was completed, the inflammation level and DNA damage of the lung tissue were detected. The specific method was as follows: lung tissue of 3 mice was randomly selected from each group, and approximately 20-30 mg of lung tissue was taken from each mouse in a centrifuge tube. Total RNA was isolated and extracted using TRIzol reagent (Invitrogen) for RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection, and the RNA was reverse transcribed into cDNA using a reverse transcription kit from Novozymes. The reaction system was: 42°C, 2 min, 37°C, 15 min, 85°C, 5 s, and placed in a -2°C or -80°C refrigerator for use, and amplified by PCR for 30 to 35 cycles. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control. PCR detection was performed using AceQ qPCR SYBR Green Master Mix fluorescent quantitative PCR reagent from Novezan and a Thermo Fisher Scientific 7900HT high-throughput rapid real-time fluorescent quantitative PCR instrument. The data were analyzed using the ΔΔCt method. The results are shown in Figure 29. The specific conditions are as follows:

[0238] Primer sequences:

[0239] GAPDH:Forward:5'-CATCACTGCCACCCAGAAGACTG-3';

[0240] Reverse:5'-ATGCCAGTGAGCTTCCCGTTCAG-3';

[0241] IL-10:Forward:5'-CGGGAAGACAATAACTGCACCC-3';

[0242] Reverse:5'-CGGTTAGCAGTATGTTGTCCAGC-3';

[0243] TNF-α:Forward:5'-GGTGCCTATGTCTCAGCCTCTT-3';

[0244] Reverse:5'-GCCATAGAACTGATGAGAGGGAG-3';

[0245] IL-1β:Forward:5'-TGGACCTTCCAGGATGAGGACA-3';

[0246] Reverse:5'-GTTCATCTCGGAGCCTGTAGTG-3';

[0247] TGF-β1:Forward:5'-TGATACGCCTGAGTGGCTGTCT-3';

[0248] Reverse:5'-CACAAGAGCAGTGAGCGCTGAA-3'.

[0249] Amplification system:

[0250] Amplification conditions:

[0251] As shown in Figure 29, the RT-PCR results showed that the mRNA level of the anti-inflammatory factor IL-10 in the X-ray group was 0.38 times that of the control group (P < 0.05), and that in the JAC4 + X-ray group was 2.91 times that of the X-ray group (P < 0.05) (Figure 29A); the mRNA level of the pro-inflammatory factor TNF-α in the X-ray group was 1.33 times that of the control group (P < 0.05), and that in the JAC4 + X-ray group was 0.63 times that of the X-ray group (P < 0.01) (Figure 29B); the mRNA level of the pro-inflammatory factor TGF-β1 in the X-ray group was 1.38 times that of the control group (P <0.05), while the JAC4+X-ray group was 0.71-fold higher than the X-ray group (P<0.01) (Figure 29C). The mRNA level of the proinflammatory cytokine IL-1β in the X-ray group was 1.90-fold higher than that in the control group (P<0.001), and in the JAC4+X-ray group was 0.43-fold higher than that in the X-ray group (P<0.001) (Figure 29D). In addition, HE staining of lung tissues in each group showed that the X-ray-treated group had thickened interstitial tissue, smaller alveolar spaces, and infiltrated a large number of inflammatory cells, while JAC4 combined with X-ray treatment thinned the alveolar walls and reduced inflammatory cell infiltration (Figure 29E). These results indicate that JAC4 alleviates X-ray-induced inflammatory responses.

[0252] To verify the protective effect of JAC4 against X-ray-induced DNA damage in lung tissue, lung tissues from three mice were randomly selected from each group for immunofluorescence staining. Proteins were extracted from lung tissues from another three mice for western blotting to measure JWA, γ-H2AX, and p-p65 expression levels. This validates the relationship between JAC4 targets and the reduction of DNA damage and apoptosis in lung tissue. The western blotting protocol is detailed in Example 4. The immunofluorescence staining protocol is as follows:

[0253] 1. Dewaxing and hydration: Place the prepared paraffin sections in a 60℃ oven for dewaxing for 2-3 hours to make the sections adhere more tightly to the glass; for dewaxing, soak the sections in xylene I for 30 minutes → xylene II for 30 minutes → xylene III for 30 minutes → anhydrous ethanol I on a shaker for 5 minutes → anhydrous ethanol II on a shaker for 5 minutes → 95% ethanol on a shaker for 5 minutes → 85% ethanol on a shaker for 5 minutes → 75% ethanol on a shaker for 5 minutes → PBS on a shaker for 5 minutes × 3.

[0254] 2. Antigen retrieval: Place the washed slides in 500 mL of 1× antigen retrieval solution (25 mL of 20× sodium citrate buffer, pH 6.0, then add 475 mL of PBS, diluted to 1×) and bring to a boil. After boiling for 10 minutes, remove the slides and allow them to cool to room temperature.

[0255] 3. Wash and block: Wash with PBS on a shaker for 5 min x 3. After washing, remove the slices and shake off the moisture from the slices, but avoid drying the slices. Use an immunofluorescent pen to draw a circle around the edge of the slice, add blocking solution (12,000g, centrifuge for 5 min) to completely cover the slice, and block at room temperature for 1 h.

[0256] 4. Wash and apply primary antibody; after removing the blocking solution, wash with PBS on a shaker for 5 min × 3; add the corresponding primary antibody (6,000g, centrifuge for 5 min) to cover the slices, and place them in a humidified box at 4°C overnight.

[0257] 5. Wash and apply secondary antibody; wash with PBS on a shaker for 5 min × 3; add corresponding fluorescent secondary antibody (6,000g, centrifuge for 5 min) and incubate at room temperature for 1 h in the dark.

[0258] 6. Wash, stain the nuclei, and photograph the slides; protect from light throughout the process and wash with PBS on a shaker for 5 minutes × 3; add anti-fluorescence quenching mounting medium containing DAPI and cover with a coverslip to seal the slide. Be careful to cover slowly from one side to avoid bubbles; place the slide in a humidified chamber after 15 minutes of sealing and continuously acquire images under a laser confocal microscope.

[0259] As shown in the immunofluorescence results of Figure 30A, compared with the control group, the JAC4, X-ray, and JAC4+X-ray groups had higher JWA fluorescence intensities. The fluorescence intensities of γ-H2AX and the inflammatory factors TNF-α and TGF-β1 in the X-ray group were significantly increased. Compared with the X-ray group, the fluorescence intensities of γ-H2AX, TNF-α, and TGF-β1 in the JAC4 combined with X-ray group were significantly decreased. Lung tissue proteins were extracted from each group. The immunoblotting results of Figures 30B-E showed that the protein levels of p-p65 in the JAC4 and X-ray groups were 0.63 times (P < 0.01) and 1.53 times (P < 0.001) higher than those in the control group, respectively, and 0.61 times (P < 0.0001) higher in the JAC4+X-ray group. The protein levels of JWA in the JAC4 and X-ray groups were 4.2 times (P < 0.0001) higher than those in the control group. The JAC4+X-ray group showed a 3-fold increase (P < 0.0001) and a 3.11-fold increase (P < 0.0001) in the expression of JWA, and the JAC4+X-ray group showed a 1.25-fold increase (P < 0.01) in the expression of X-ray. The γ-H2AX protein levels in the JAC4 and X-ray groups were 0.91-fold (P = 0.90) and 2.68-fold (P < 0.0001) higher than those in the control group, respectively, and the JAC4+X-ray group showed a 0.83-fold increase (P < 0.05) in the expression of X-ray. These results were consistent with those of the immunofluorescence assay. These results suggest that JAC4 combined with X-ray promotes JWA expression and inhibits p-p65, further alleviating X-ray-induced DNA damage in lung tissue.

[0260] Example 22 Effects of JAC4 combined with X-ray radiation on human lung adenocarcinoma cells SPCA-1 and human normal lung epithelial cells BEAS-2B

[0261] SPCA-1 and BEAS-2B cells were cultured in 90% DMEM + 10% FBS + 100 μg / ml ciprofloxacin. SPCA-1 and BEAS-2B cells were pretreated with 10 μM JAC4 for 24 hours. CCK-8 assay was performed 24 hours after 4 Gy X-ray treatment to explore the effect of JAC4 combined with X-ray on the viability of SPCA-1 and BEAS-2B cells. The specific method was as follows:

[0262] 1. SPCA-1 and BEAS-2B cells in the exponential growth phase were cultured in 96-well plates at a density of 5,000 cells per well. Three replicates were set up for each group.

[0263] 2. After 24 h of culture, when the cells were completely attached and restored to normal morphology, they were irradiated with 0, 4, 8, 12, 16, or 20 Gy of X-ray or treated with 0, 1, 5, 10, 20, or 50 μM of JAC4.

[0264] 3. After 24 hours of treatment, add 10 μL of CCK-8 reagent to each well, incubate in a cell culture incubator for 1 hour, and measure the absorbance (OD) at a wavelength of 450 nm.

[0265] 4. Cell viability calculation formula: Cell viability (%) = (OD 对照组 -OD 处理组 )×100% / (OD 对照组 -OD 空白孔 ).

[0266] The results are shown in Figure 31. After X-ray treatment, the viability of SPCA-1 and BEAS-2B cells was reduced. JAC4 combined with X-ray significantly inhibited the viability of SPCA-1 cells (Figure 31A); while JAC4 combined with X-ray alleviated the inhibitory effect of X-ray on the viability of BEAS-2B cells (Figure 31B).

[0267] The cell proliferation ability is reflected by plate colony formation test. The specific method is as follows:

[0268] 1. Take exponentially growing SPCA-1 and BEAS-2B cells (or SPCA-1 and BEAS-2B cells transfected with JWA knockdown plasmid) and plate them in 6-well plates at a specific number of cells. The specific number of cells plated is as follows

[0269] 2. After cell attachment, treat with or without 10 μM JAC4 and then expose to 0, 2, 4, or 6 Gy of X-rays. Replace the culture medium with the corresponding concentration of JAC4 every 2 days (or after cell attachment, expose the cells to 0, 2, 4, or 6 Gy of X-rays and replace the culture medium with fresh medium every 2 days).

[0270] 3. After 10-14 days of cell culture, visible cell clones appear. Discard the culture medium, fix the cells with methanol for 10-20 minutes, stain with crystal violet for 10 minutes, and then slowly rinse with running water three times. Take pictures and count the number of clones with >50 colony-forming cells.

[0271] 4. Calculate the colony forming efficiency (CFE) = (number of colonies formed / number of cells seeded) × 100%. Then calculate the cell survival fraction (SF) based on the colony forming efficiency = (colony forming efficiency of each dose group / colony forming efficiency of the 0 Gy dose group) × 100%.

[0272] The results showed that JAC4 combined with X-ray treatment reduced the number of SPCA-1 cell colonies (Figure 31C) but increased the number of BEAS-2B cell colonies (Figure 31E). Using a single-target multi-target model to fit the radiosensitization curves, 10 μM JAC4 combined with X-ray significantly reduced SPCA-1 cell survival compared to the X-ray alone group (Figure 31D), while 10 μM JAC4 combined with X-ray slightly increased BEAS-2B cell survival compared to the X-ray alone group (Figure 31F). These results suggest that JAC4 combined with X-ray inhibits the viability and proliferation of SPCA-1 cells and increases the viability and proliferation of BEAS-2B cells.

[0273] To investigate the effects of JAC4 combined with X-ray on DSBs in SPCA-1 and BEAS-2B cells, SPCA-1 and BEAS-2B cells were pretreated with 10 μM JAC4 for 24 hours. Two hours after 4 Gy X-ray treatment, γ-H2AX fluorescence labeling and Western blotting were performed (see Example 4 for details). The results are shown in Figure 32. Compared with the control group, numerous γ-H2AX foci appeared in the nuclei of both SPCA-1 and BEAS-2B cells after 4 Gy X-ray irradiation. Compared with the X-ray alone treatment group, JAC4 combined with X-ray treatment resulted in an increase in γ-H2AX foci in SPCA-1 cells (Figures 32A, C). In BEAS-2B cells, JAC4 combined with X-ray significantly reduced γ-H2AX foci formation in BEAS-2B cells compared with the X-ray treatment group (Figures 32B, D). Western blotting results showed that 4 Gy X-ray irradiation increased γ-H2AX expression compared to the control group. Compared to the X-ray alone group, JAC4 combined with X-ray treatment increased JWA expression in SPCA-1 cells and also increased γ-H2AX expression (Figure 32E). In BEAS-2B cells, JAC4 combined with X-ray increased JWA expression while decreasing γ-H2AX protein expression (Figure 32F). These results suggest that JAC4 combined with X-ray increases X-ray-induced DSBs in SPCA-1 cells by activating JWA expression and mitigates DSBs in BEAS-2B cells.

[0274] Severe DSBs can lead to apoptosis. SPCA-1 and BEAS-2B cells were plated and cultured for 24 hours. After treatment with 10 μM JAC4 for 24 hours, the cells were treated with 4 Gy X-ray for 24 hours. Hoechst 33342 staining was performed (see Example 13 for details on the Hoechst staining method for detecting cell apoptosis). The effects of JAC4 combined with irradiation on apoptosis were evaluated. The results are shown in Figure 33. Compared with the control group, X-ray increased apoptosis in SPCA-1 cells, and JAC4 enhanced the pro-apoptotic effect of X-ray on SPCA-1 cells (Figures 33A, B). In contrast, in BEAS-2B cells, X-ray increased apoptosis in BEAS-2B cells compared with the control group, while JAC4 reduced the pro-apoptotic effect of X-ray on BEAS-2 cells (Figures 33A, C). A decrease in mitochondrial membrane potential (JC-1) is a hallmark of early apoptosis. Mitochondrial membrane potential was measured using the fluorescent probe JC-1. Red fluorescence indicates the formation of JC-1 aggregates due to high mitochondrial membrane potential, while green fluorescence indicates depolarization of mitochondrial membrane potential. The ratio of red to green fluorescence represents the mitochondrial membrane potential and the number of early apoptotic cells. Mitochondrial membrane potential assays showed that X-ray treatment caused a decrease in mitochondrial membrane potential, indicating an increase in the number of early apoptotic cells. JAC4 combined with X-ray treatment exacerbated the decrease in mitochondrial membrane potential in SPCA-1 cells (Figure 33D, E). Conversely, JAC4 reversed the X-ray-induced decrease in mitochondrial membrane potential in BEAS-2B cells (Figure 33D, F).

[0275] X-ray-induced production of large amounts of reactive oxygen species (ROS) and sustained oxidative stress can cause DNA damage. Superoxide dismutase (SOD) and catalase (CAT) exert antioxidant effects, while lipid oxidation (MDA) and ROS can reflect the level of oxidative stress in vivo. The fluorescent probe DCFH-DA was used to detect intracellular ROS levels. The ROS level detection experimental results in Figure 34 show that X-ray increases intracellular ROS levels, while JAC4 combined with X-ray significantly reduces ROS levels in BEAS-2B cells. However, JAC4 had no significant effect on ROS levels in SPCA-1 cells. The supernatant of BEAS-2B cells was extracted for MDA level detection, CAT and SOD activity detection test. The results showed that the SOD activities of BEAS-2B cells in DMSO, JAC4, X-ray and JAC4+X-ray groups were 30.00±0.14, 28.67±1.36, 21.39±0.79 and 24.20±1.18 U / mg, respectively (P<0.0001), and the CAT activities were The levels of the antioxidant product MDA were 3.45±0.16, 3.42±0.08, 2.11±0.15, and 3.56±0.02 U / mg (P<0.0001), respectively. The levels of the oxidative product MDA were 23.03±2.54, 24.95±1.63, 48.10±3.73, and 31.35±4.34 μM / mg (P<0.0001), respectively. This restored the antioxidant capacity of BEAS-2B cells and reduced the production of oxidative products. These results suggest that JAC4 reduces X-ray-induced DNA damage and apoptosis in BEAS-2B cells by increasing the activities of SOD and CAT antioxidant enzymes and reducing the production of MDA and ROS.

[0276] ROS production triggers NF-κB activation, promoting its translocation to the nucleus and mediating various inflammatory responses. Nuclear and cytoplasmic protein separation was performed on BEAS-2B cells in each group. Western blotting results (Figure 35) show that NF-κB expression in the cytoplasm remained largely unchanged in all groups. However, nuclear expression increased after X-ray treatment. However, JAC4 combined with X-ray treatment inhibited this X-ray-induced increase in nuclear NF-κB expression. Figure 35 shows that X-ray can induce NF-κB translocation from the cytoplasm to the nucleus, and JAC4 combined with X-ray prevented this translocation and exerted its effects. These results demonstrate that JAC4 inhibits NF-κB nuclear translocation, which mediates inflammatory responses.

[0277] To clarify that JAC4 exerts its synergistic and attenuating effects in SPCA-1 and BEAS-2B cells by activating JWA and combining with X-ray, the JWA knockdown plasmid was transfected into SPCA-1 and BEAS-2B cells.

[0278] As shown in Figure 36, the results of the plate colony formation assay showed that SPCA-1 cells transfected with the JWA knockdown plasmid formed significantly more colonies after JAC4 treatment compared to the non-knockdown si-Control group. Using a single-target model to fit the radiosensitization curve, the survival rate of SPCA-1 cells transfected with the JWA knockdown plasmid treated with JAC4 was significantly increased compared to the JAC4-treated si-Control group. The survival rate of BEAS-2B cells transfected with the JWA knockdown plasmid treated with JAC4 was significantly decreased compared to the non-knockdown si-Control group treated with JAC4. These results further confirm that JAC4 plays a role in enhancing and reducing toxicity in X-ray therapy for NSCLC by activating JWA. As shown in Figure 37, after 48 hours of transfection of the JWA knockdown plasmid into SPCA-1 and BEAS-2B cells, X-ray treatment was performed 2 hours later. The immunofluorescence experimental results showed that compared with the si-Control control group, the nuclear γ-H2AX fluorescence intensity and γ-H2AX protein expression of SPCA-1 cells transfected with the si-Control plasmid were significantly increased after irradiation. Compared with the irradiated SPCA-1 cells transfected with the si-Control plasmid, the γ-H2AX fluorescence intensity of cells with JWA knockdown plasmid was significantly increased after irradiation. The results showed that JWA plays a significant role in tumor cell killing. Compared with the si-Control control group, the nuclear γ-H2AX fluorescence intensity and γ-H2AX protein expression of BEAS-2B cells transfected with the si-Control plasmid were significantly increased after irradiation. Compared with the irradiated SPCA-1 cells transfected with the si-Control plasmid, the γ-H2AX fluorescence intensity and γ-H2AX protein expression of cells knocked down with the JWA plasmid were higher after irradiation, reflecting the importance of JWA in protecting normal cells. These results indicate that the loss of JWA weakens the synergistic and attenuating effects of X-ray in SPCA-1 and BEAS-2B cells.

[0279] In summary, this disclosure demonstrates that JAC4 differentially regulates X-ray-induced DNA double-strand breaks and apoptosis in SPCA-1 and BEAS-2B cells. In SPCA-1 cells, JAC4 activation of JWA increased X-ray-induced γ-H2AX and apoptotic cell counts, whereas in BEAS-2B cells, γ-H2AX and apoptotic cell counts were significantly reduced. JAC4 must activate JWA to exert its synergistic and attenuating effects on X-ray irradiation. Compared with the NC control group, the colony formation rate of SPCA-1 cells transfected with JWA knockdown was significantly increased, whereas the colony formation rate of BEAS-2B cells transfected with JWA knockdown was significantly decreased. Compared with the NC control group, the number of DSBs and apoptotic cell counts in SPCA-1 cells transfected with JWA knockdown was reduced, whereas the number of DSBs and apoptotic cell counts in BEAS-2B cells transfected with JWA knockdown was increased. Furthermore, JAC4, in combination with X-ray, exerted a synergistic and attenuating effect in a subcutaneous NSCLC tumor-bearing mouse model. In the subcutaneous tumor-bearing model, the tumor inhibition rate of the JAC4+X-ray group was significantly higher than that of the X-ray alone treatment group; in addition, the combined group significantly reduced the infiltration of inflammatory cells in the lung tissue, increased the anti-inflammatory factor IL-10, and reduced the mRNA levels of the pro-inflammatory factors IL-1β, TNF-α and TGF-β1.

[0280] Example 23 R-JAC4 reduces X-ray radiation-induced damage to intestinal epithelial cell monolayer integrity

[0281] Caco-2 cells were cultured in 78% MEM + 20% FBS + 1% P / S + 1% NEAA. Caco-2 cells were seeded at a density of 10,000 cells / well in a 24-well chamber. The culture medium was changed every 2 days for 11 days, and then pretreated with DMSO or 1, 3, or 10 μM R-JAC4 for 3 days. The monolayer membrane model was constructed and matured for a total of 14 days. It was irradiated with a dose of 0 Gy or 20 Gy at a radiation rate of 2.5 Gy / min. The monolayer membrane resistance and FD4 penetration were detected after 24 hours. The results are shown in Figure 38.

[0282] The results showed that the resistance of Caco-2 monolayer in the irradiated group was significantly reduced compared with that in the DMSO group (960.70±67.94 vs 1189±128.9Ω·cm 2), FD4 permeation increased significantly (678.90±91.26vs 280.3±37.40ng / ml), indicating that the integrity of the monolayer membrane was destroyed after 20Gy irradiation; compared with the irradiation group, 1, 3, and 10μM R-JAC4 significantly improved the FD4 permeation of the monolayer membrane after irradiation (410.60±50.99, 345.40±29.85, 377.30±48.24ng / ml) and alleviated the decrease in resistance (1016±64.65, 1138±25.97, 1109±101.20Ω·cm 2 ), demonstrating that R-JAC4 has a protective effect against irradiation-induced damage to the Caco-2 monolayer integrity.

Claims

1. Use of a JWA gene agonist or a pharmaceutical composition thereof in preventing or treating radiation damage.

2. The use according to claim 1, characterized in that The radiation injury is selected from one or both of radiation intestinal injury and radiation lung injury.

3. The use according to claim 2, characterized in that The radiation injury is radiation-induced intestinal injury.

4. The use according to claim 2 or 3, characterized in that The radiation-induced intestinal injury is radiation enteritis.

5. The use according to claim 2, characterized in that The radiation injury is radiation-induced lung injury.

6. The use according to claim 2 or 5, characterized in that The radiation lung injury is radiation pneumonitis.

7. The use according to any one of claims 1 to 6, characterized in that The radiation damage is selected from one or both of radiation oxidative stress damage and radiation DNA damage.

8. The use according to claim 1, characterized in that The radiation damage is selected from X-ray radiation damage.

9. A method for preventing or treating radiation damage, characterized in that: The method comprises administering a therapeutically effective amount of a JWA gene agonist or a pharmaceutical composition thereof to a mammal, preferably a human, in need of such treatment.

10. Use of a JWA gene agonist or a pharmaceutical composition thereof in the preparation of a drug for preventing or treating radiation damage.

11. A JWA gene agonist or a pharmaceutical composition thereof for preventing or treating radiation damage.

12. Use of a JWA gene agonist or a pharmaceutical composition thereof in the preparation of a drug for preventing or treating cancer patients, wherein the drug is administered in combination with radiotherapy.

13. A method for preventing or treating cancer in a mammal, comprising administering a JWA gene agonist or a pharmaceutical composition thereof and radiotherapy to a mammal, preferably a human, in need of such treatment.

14. Use of a JWA gene agonist or a pharmaceutical composition thereof in combination with radiotherapy in the prevention or treatment of cancer.

15. A combination of a JWA gene agonist or a pharmaceutical composition thereof and radiotherapy for preventing or treating cancer.

16. The use, method, composition or combination according to any one of claims 12 to 15, wherein the cancer is selected from lung cancer.

17. The use, method, composition or combination according to claim 16, wherein the cancer is selected from non-small cell lung cancer.

18. The use, method, composition or combination according to claim 17, wherein the cancer is selected from lung adenocarcinoma.

19. The use, method, composition or combination according to any one of claims 1 to 18, characterized in that The JWA gene agonist is selected from the compound of formula (I) or a pharmaceutically acceptable salt thereof, 20. The use, method, composition or combination according to claim 19, characterized in that The compound of formula (I) is selected from One or a combination of two.

21. The use, method, composition or combination according to claim 19, characterized in that The compound of formula (I) is selected from 22. The use, method, composition or combination according to any one of claims 1 to 21, characterized in that The pharmaceutical composition comprises the JWA gene agonist and pharmaceutically acceptable excipients.

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