Application of Phoximus in preparation of drugs for reversing gastric cancer drug resistance

Abstract

The application discloses application of Ophisaurus apodus in preparation of a drug for reversing drug resistance of gastric cancer and belongs to the technical field of medicines. The application can reduce tumor volume and size, promote drug resistance cell apoptosis, and reverse the chemotherapeutic drug resistance of a nude mouse with gastric cancer by jointly using Ophisaurus apodus in different parts of the body and cisplatin, by down-regulating P-gp, MRP1 and Bcl-2 protein expression, and by up-regulating Bax protein expression. The effect of the tail + cisplatin group is the most significant.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to the application of the spotted lizard in the preparation of drugs to reverse drug resistance in gastric cancer. Background Technology

[0002] Gastric cancer is one of the most common malignant tumors of the digestive tract, ranking fifth in incidence and fourth in mortality worldwide, seriously threatening human health. Due to the lack of obvious symptoms and specific biomarkers for early diagnosis, most patients are diagnosed at an advanced stage, missing the optimal time for surgery. Chemotherapy is one of the main treatment methods for patients with advanced gastric cancer. Cisplatin (DDP) drugs are the main drugs for preoperative chemotherapy, palliative treatment, and advanced and metastatic gastric cancer. They can inhibit the replication of cancer cell DNA and destroy their cell membrane structure, exhibiting a strong broad-spectrum anti-cancer effect. However, with prolonged use, varying degrees of drug resistance easily develop, leading to decreased sensitivity to chemotherapy and poor clinical efficacy. Therefore, exploring drugs with significant effects and few side effects, and improving the sensitivity of tumor cells to chemotherapy drugs, is of great importance and urgency. In particular, exploring traditional Chinese medicine with characteristics such as safety, low toxicity, multiple targets, and multiple pathways has become a hot topic in the treatment of gastric cancer, and is also of great significance to the quality of life of patients.

[0003] *Spotted Lizard* is the dried whole body of a species in the genus *Spotted Lizard* of the family Lacertidae. First recorded in the *Shennong Bencao Jing* (Shennong's Classic of Materia Medica), it is salty and cold in nature, and enters the lung, liver, and kidney meridians. The *Shennong Bencao Jing* records the lizard's effects as "treating five types of urinary retention due to pathogenic factors, breaking up urinary stones, treating hematuria, and promoting urination," possessing the functions of promoting blood circulation, removing blood stasis, eliminating goiter and nodules, promoting urination, and calming the nerves. Studies have shown that *Spotted Lizard* is rich in protein, amino acids, and various trace elements, and its tail has strong regenerative ability, exhibiting good anti-tumor effects and providing good protection for the body. However, there are no reports on whether *Spotted Lizard* can reverse cisplatin resistance in gastric cancer. Therefore, this invention provides the application of *Spotted Lizard* in the preparation of drugs to reverse gastric cancer drug resistance. Summary of the Invention

[0004] This invention provides the application of the spotted lizard in the preparation of drugs to reverse drug resistance in gastric cancer.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] This invention provides the application of the spotted lizard in the preparation of drugs to reverse drug resistance in gastric cancer.

[0007] Preferably, the drug for reversing gastric cancer drug resistance refers to the drug that reverses the resistance of gastric cancer to cisplatin-based drugs.

[0008] Preferably, the cisplatin-based drugs include cisplatin, carboplatin, oxaliplatin, and lobaplatin.

[0009] Preferably, the use of the spotted lizard in the preparation of drugs that downregulate the expression of P-gp, MRP1 and Bcl-2 proteins.

[0010] Preferably, the use of the spotted lizard in the preparation of a drug that upregulates Bax protein expression.

[0011] Preferably, the drug is in solid, semi-solid, or liquid form.

[0012] Preferably, the formulation of the drug includes aqueous solution, non-aqueous solution, suspension, lozenge, capsule, tablet, granule, pill or powder.

[0013] Preferably, the drug is administered via injection or oral administration.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0015] The application of *Lithops nigra* (a type of lizard) in the preparation of drugs to reverse drug resistance in gastric cancer, as provided in this invention, showed that compared with the model group, the tumor weight in each treatment group was reduced (P<0.05, P<0.01), the expression of P-gp, MRP1, and Bcl-2 proteins was significantly decreased in each treatment group (P<0.05), the expression of Bax protein was significantly increased (P<0.05), and the apoptosis rate of drug-resistant cells was significantly increased (P<0.05). Compared with the cisplatin group, the tumor weight in each group of *Lithops nigra* from different tumor sites plus cisplatin was significantly reduced (P<0.01). 5) The tumor weight reduction was most significant in the Ningxia spotted lizard tail + cisplatin group (P<0.05), with the highest inhibition rate; the expression of P-gp, MRP-1, and Bcl-2 proteins was decreased (P<0.05), while the expression of Bax protein was increased (P<0.05), with the former two being most significant in the Ningxia spotted lizard tail + cisplatin group (P<0.05); the apoptosis rate of drug-resistant cells in nude mouse tumor tissues was significantly increased in all groups (P<0.01), with the most significant increase in apoptosis rate in the Ningxia spotted lizard tail + cisplatin group (P<0.05). Different parts of the Ningxia spotted lizard combined with cisplatin all had varying degrees of effect in reversing cisplatin resistance in nude mice with gastric cancer, with the tail + cisplatin group showing the most significant sensitization and efficacy enhancement effect. Attached Figure Description

[0016] Figure 1 This invention provides a comparison of tumor size in nude mice.

[0017] Figure 2 The expression of P-gp, MRP1, Bcl-2 and Bax proteins in nude mouse tumor tissues by Western blotting method of this invention;

[0018] Figure 3 The expression of P-gp, MRP1, Bcl-2, and Bax in nude mouse tumor tissues according to the present invention;

[0019] Figure 4 This is the apoptosis situation of drug-resistant cells in the tumor tissue of nude mice in the present invention. Detailed implementation manners

[0020] In order to enable those skilled in the art to better understand and implement the technical solution of the present invention, the present invention will be further described below in conjunction with specific embodiments and drawings. However, the provided embodiments are not intended to limit the present invention. The following test methods and detection methods are all conventional methods unless otherwise specified; the reagents and raw materials are all commercially available unless otherwise specified.

[0021] Embodiment

[0022] Materials and instruments

[0023] Animals: 36 male BALB / c nude mice, 4 - 6 weeks old, SPF grade, body weight 18 - 22 g; purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., license number: SCXK (Beijing) 2021 - 0006; raised in the Experimental Animal Center of Ningxia Medical University, with free access to food and water. Experiments were carried out after 3 days of adaptive feeding. The animal experiment was approved by the Ethics Review Committee of Ningxia Medical University.

[0024] Cell line: Human gastric adenocarcinoma MKN - 45 / DDP drug-resistant cell line, purchased from Zhejiang Misen Cell Technology Co., Ltd.

[0025] Main drugs and reagents

[0026] Eremias multiocellata purchased from the outpatient department of the Affiliated Traditional Chinese Medicine Hospital of Ningxia Medical University (batch number: 2208036), and decocted into a traditional Chinese medicine decoction with a concentration of 0.2 g / ml; cisplatin (purity ≥99.9%, specification: 100 mg / vial, batch number: 124L021, Beijing Solarbio Science & Technology Co., Ltd.).

[0027] Hematoxylin staining solution (batch number: BA-4041, Zhuhai Beso Biotechnology Co., Ltd.), differentiation medium (C0163M, Shanghai Beyotime Biotechnology Co., Ltd.), total protein extraction kit, BCA protein content detection kit (batch number: 20220311, KGPBCA, Jiangsu Kaiji Biotechnology Co., Ltd.); RPMI 1640 culture medium, penicillin-streptomycin mixture (batch numbers: 2211068, 20220705, Shanghai Datashire Biotechnology Co., Ltd.), fetal bovine serum (batch number: 2222092, Beijing Solarbio Science & Technology Co., Ltd.); Anti-P-gp (batch number: 25081-1-ap, Wuhan Sanying), Anti-MRP1, rabbit-derived Bcl-2 related X protein monoclonal antibody (Bax), β-actin (batch numbers: DF7148, AF0120, AF7018, Affinity), rabbit-derived B-cell lymphoma 2 gene (Bcl-2) polyclonal antibody (batch number: BS0032R, BIOSS); HRP-labeled goat anti-rabbit, HRP-labeled goat anti-mouse (batch numbers: ZB2301, ZB2305, Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.), protein-free rapid blocking solution (batch number: 03631200, Shanghai Yamei Biotechnology Co., Ltd.).

[0028] Main instruments

[0029] Cell culture incubator (Thermo Fisher Scientific, USA), EVOS X1 core cell imaging system inverted biological microscope, Arcadia H+C tissue paraffin embedding machine (including freezing stage), TP1020 fully automatic tissue dehydrator, RM2255 fully automatic rotary paraffin microtome, HI1210 slide burner, HI1220 slide spreader, Aperio LV1 slide scanner (Germany / Lei-ca), JXFSTPRP-CL cryogenic grinder (Shanghai Jingxin), A-14C small high-speed refrigerated centrifuge, Amersham 1mager 680RGB ultrasensitive multi-functional imager (USA / GE), Mini Protean Tetra protein electrophoresis system (Bio-Rad Laboratories, USA).

[0030] Test methods

[0031] Establishment and grouping of MKN-45 / DDP gastric cancer subcutaneous tumor cisplatin resistance model in nude mice

[0032] Cell culture: MKN-45 / DDP cells were cultured in RPMI1640 complete medium containing 0.1ug / ml cisplatin (DDP) to maintain their drug resistance. Two weeks before modeling, the medium was changed to cisplatin-free complete medium and cultured every 2-3 days. The cells were then placed in a cell culture incubator at 37℃ with 5% CO2 for routine culture.

[0033] Preparation of aqueous extract of the spotted lizard: The head, limbs, trunk and tail of the dried spotted lizard were ground into fine powder, put into small bags, and boiled twice in 1000ml of distilled water. The supernatants from the two extractions were combined and concentrated to a crude drug concentration of 0.4g / ml. The extract was then stored at 4℃ for later use.

[0034] Inoculation: MKN-45 / DDP drug-resistant gastric cancer cells in logarithmic growth phase were harvested and their concentration adjusted to 1×10⁻⁶ cells / mL with PBS. 7 Prepare a cell suspension of 0.2 ml / cell for later use. Under aseptic conditions, slowly inject 0.2 ml of MKN-45 / DDP single-cell suspension subcutaneously into the right anterior axillary region of each nude mouse. After cell inoculation, observe and weigh the nude mice daily. Observe tumor formation in the nude mice approximately 7 days after injection. When the tumor volume reaches approximately 4 mm in diameter, the subcutaneous tumor model in nude mice is considered successful.

[0035] Grouping: After successful modeling, the experimental nude mice were randomly divided into 6 groups: model group, cisplatin group, Ningxia spotted lizard head + cisplatin group, Ningxia spotted lizard limbs + cisplatin group, Ningxia spotted lizard trunk + cisplatin group, and Ningxia spotted lizard tail + cisplatin group.

[0036] Administration: Drug administration began when the tumor reached 6 mm in length. The dosage was calculated based on the commonly used clinical dosage, adult daily dosage, and mouse body surface area to determine the equivalent dose for nude mice. The model group was administered 0.9% saline by gavage (0.1 ml / 10 g), twice daily for 28 days; the cisplatin group was administered cisplatin intraperitoneally, 2 mg / kg, twice weekly for 8 weeks; the Ningxia spotted lizard head, limbs, trunk, and tail + cisplatin groups were administered the corresponding water extract decoction of the spotted lizard head, limbs, trunk, and tail by gavage (0.1 ml / 10 g), twice daily for 28 days, and all groups were simultaneously administered cisplatin intraperitoneally (2 mg / kg), twice weekly for 8 weeks.

[0037] Tumor weight and tumor inhibition rate measurement

[0038] After grouped drug intervention, the weight of nude mice was measured daily, and the tumor volume was measured every 3-4 days. The nude mice were sacrificed 24 hours after the last drug administration, and the tumor was aseptically and completely dissected, the surrounding fibrous capsule was removed, and the tumor tissue weight of each group of nude mice was weighed. The tumor inhibition rate was calculated based on the tumor weight: Tumor inhibition rate (%) = (1 - average tumor weight of the drug administration group / average tumor weight of the model group) × 100%.

[0039] Immunoblotting was used to detect the expression of drug resistance-related proteins P-gp, MRP1, Bcl-2, and Bax in tumor tissues of nude mice in each group.

[0040] Total protein was extracted from tumor tissues treated with water extracts and cisplatin from different parts of the lizard *Triplophysa pulcherrima*. Protein concentration was determined using a BCA protein assay kit. The extracted protein solution was added to 5X reducing protein loading buffer at a ratio of 4:1, denatured in a boiling water bath for 10 min, and then refrigerated at -20°C for later use. During the experiment, 8 μg of protein sample was added to each well. After electrophoresis to achieve protein separation at various molecular weights, the sample was transferred to a PVDF membrane, blocked with rapid blocking buffer for 15 min, washed 5 times, and then P-gp (1:1000), MRP1 (1:1200), Bcl-2 (1:1000), and Bax (1:800) were added. The membrane was incubated overnight at 4°C. The next day, after warming and washing, secondary antibody (1:4000) was added, and the membrane was incubated at room temperature for 2 h. After washing 5 times, images were acquired, and the gray values ​​of P-gp, MRP1, Bcl-2, Bax, and β-actin were analyzed using ImageJ software.

[0041] Immunohistochemistry was used to detect the expression of P-gp, MRP1, Bcl-2 and Bax proteins in tumor tissues of nude mice in each group.

[0042] Sections were incubated overnight at 4°C, dewaxed to water as usual, and then autoclaved in 3% citric acid retrieval solution for 10 min. After blocking, primary and secondary antibodies were added for incubation. The sections were counterstained with DAB and hematoxylin, mounted, and observed under a microscope for image acquisition. Positive expression regions were selected based on the images of positive immunoreactions, and ImageJ software was used to analyze the positive results. The expression level was expressed as integrated optical density / total image area.

[0043] TUNEL assay was used to detect apoptosis of drug-resistant cells in tumor tissues of nude mice in each group.

[0044] Routine sectioning, baking, dewaxing, and hydration were performed. Tumor tissue was treated with Proteinase K working solution at 37°C for 20 min, followed by rinsing three times. Each tissue was then treated with 50 μL of TUNEL reaction mixture at 37°C for 1 h, followed by rinsing three times. DAPI staining solution was added to the samples, and the mixture was incubated for 20 min, followed by rinsing three times. The samples were mounted, and finally observed and photographed using a fluorescence microscope at a green wavelength of 488 nm. Apoptotic cell nuclei showed green fluorescence, while DAPI-stained cell nuclei showed blue fluorescence. The results were analyzed using ImageJ software.

[0045] Statistical methods

[0046] Data were analyzed using SPSS 25.0 statistical software, and results are expressed as mean ± standard deviation (SD). ± The data were tested for normality and homogeneity of variance. The t-test was used for comparisons between two groups, and one-way ANOVA was used for comparisons among multiple groups. p<0.05 was considered statistically significant, and p<0.01 was considered highly significant.

[0047] Test results

[0048] Tumor weight and tumor inhibition rate in each group of nude mice

[0049] Tumor weight comparison in nude mice: Compared with the model group, the tumor weight in the cisplatin group was significantly reduced (P<0.05). The tumor weight in all groups (head, limbs, trunk, and tail) of the *Symplocos ningpoensis* plus cisplatin was further reduced (P<0.01). Compared with the cisplatin group, the tumor weight in all groups (head, limbs, trunk, and tail) of the *Symplocos ningpoensis* plus cisplatin was reduced (P<0.05). Within each combined group, the tail plus cisplatin group showed the most significant reduction in tumor weight (P<0.05). Tumor inhibition rate comparison: Compared with the model group, the tumor inhibition rate increased sequentially from the cisplatin group to the head, limbs, trunk, and tail plus cisplatin groups of the *Symplocos ningpoensis*, with the tail plus cisplatin group showing the highest inhibition rate at 77%. See Table 1.

[0050] Table 1 Comparison of weight and tumor inhibition rate of nude mice in each group ( (n=6)

[0051]

[0052] Observation of tumor tissue size in each group of nude mice

[0053] The size of tumors in each group of nude mice was observed, such as... Figure 1 As shown, differences in tumor size were observed. The tumors were round or nearly round, brittle and hard, with some tumors easily ulcerated and bleeding on the surface. A thin membrane covered the surface, which was easily peeled off. Some tumors showed necrotic foci in the center after incision. The tumors in the model group were significantly larger than those in any of the treatment groups; the tumors in the cisplatin group were larger than those in different parts of the Ningxia spotted lizard + cisplatin group, with the tail + cisplatin group having the smallest tumor volume.

[0054] Expression of P-gp, MRP1, Bcl-2 and Bax proteins in tumor tissues of nude mice in each group

[0055] like Figure 2 As shown, compared with the model group, the expression of P-gp, MRP1, and Bcl-2 proteins was significantly decreased in all treatment groups (P<0.05); the expression of Bax protein was significantly increased (P<0.05). Compared with the cisplatin group, the expression of P-gp, MRP-1, and Bcl-2 proteins in tumor tissues of the head, limbs, trunk, and tail of the Ningxia spotted lizard + cisplatin group was further decreased (P<0.05), and the expression of Bax protein was further increased (P<0.05). Among the different lizard parts + cisplatin combination groups, the tail + cisplatin group of the Ningxia spotted lizard showed the most significant decrease in P-gp, MRP1, and Bcl-2 protein expression and the most significant increase in Bax protein expression (P<0.05).

[0056] Immunohistochemistry was used to detect the expression of P-gp, MRP1, Bcl-2, and Bax in tumor tissues of nude mice in each group.

[0057] In each group, the positive expression of P-gp and MRP1 proteins was mainly concentrated in the cytoplasm, with some in the cell membrane, and appeared as brownish-yellow or brown granules. They were mostly diffusely distributed in patches in the tissue field. The positive expression of Bcl-2 protein was mainly concentrated in the cytoplasm and around the nucleus, and appeared as brownish-yellow granules. The positive expression of Bax protein was mainly in the cytoplasm, and appeared as brownish-yellow granules.

[0058] Compared with the model group, the positive expression levels of P-gp, MRP1, and Bcl-2 proteins were significantly reduced in all treatment groups (P<0.05), while the positive expression level of Bax protein was significantly increased (P<0.05). Compared with the cisplatin group, the positive expression levels of P-gp, MRP-1, and Bcl-2 proteins in tumor tissues from the head, limbs, trunk, and tail of *Lithops nigra* in Ningxia were further reduced (P<0.05), while the positive expression level of Bax protein was further increased (P<0.05). Among the different sites of *Lithops nigra* treated with cisplatin, the tail of *Lithops nigra* treated with cisplatin showed the most significant reduction in the positive expression levels of P-gp, MRP1, and Bcl-2 proteins and the most significant increase in the positive expression level of Bax protein (P<0.01). Figure 3 As shown in Table 2.

[0059] Table 2. Expression of P-gp, MRP1, BCl-2, and Bax in tumor tissues of nude mice in each group ( )

[0060]

[0061] Note: *Compared with the model group, *P<0.05, **P<0.01; #Compared with the cisplatin group, #P<0.05, ##P<0.01; △Comparison within each combined group, △P<0.01

[0062] TUNEL assay was used to detect apoptosis of drug-resistant cells in gastric cancer nude mouse tumor tissues from each group.

[0063] Compared with the model group, the apoptosis rate of drug-resistant cells in tumor tissues of each treatment group was significantly increased, with statistically significant differences (P<0.05); compared with the cisplatin group, the apoptosis rate of drug-resistant cells in tumor tissues of the head, limbs, trunk, and tail of the Ningxia spotted lizard + cisplatin group was further increased (P<0.01); among the different parts of the spotted lizard + cisplatin groups, the apoptosis rate of the tail + cisplatin group was the most significantly increased (P<0.05). Figure 4 As shown in Table 3.

[0064] Table 3. Apoptosis of drug-resistant cells in tumor tissues of nude mice in each group ( )

[0065]

[0066] Note: *Compared with the model group, *P<0.05, **P<0.01; #Compared with the cisplatin group, #P<0.01; △Comparison within each combined group, △P<0.05

[0067] In summary, combining cisplatin with different parts of the body of the Ningxia spotted lizard can reduce tumor volume and size, promote apoptosis of drug-resistant cells, and reverse chemotherapy resistance in nude mice with gastric cancer by downregulating the expression of P-gp, MRP1 and Bcl-2 proteins and upregulating the expression of Bax protein. The tail + cisplatin group showed the most significant effect.

[0068] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. The application of the spotted lizard in the preparation of drugs to reverse drug resistance in gastric cancer, characterized in that, The drug that reverses drug resistance in gastric cancer refers to the drug that reverses the resistance of gastric cancer to cisplatin-based drugs.

2. The application according to claim 1, characterized in that, The cisplatin-based drugs include cisplatin, carboplatin, oxaliplatin, and lobaplatin.

3. The application according to claim 1, characterized in that, The drug is in solid, semi-solid, or liquid form.

4. The application according to claim 1, characterized in that, The formulations of the drug include aqueous solutions, non-aqueous solutions, suspensions, lozenges, capsules, tablets, granules, pills, or powders.

5. The application according to claim 1, characterized in that, The drug can be administered by injection or orally.

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