Application of GHSR antagonist in preparation of medicine for treating echinococcosis granulosa
By using the GHSR antagonist [D-Lys3]-GHRP-6, the adverse reactions and drug resistance problems of existing methods for treating echinococcosis were solved, and the inhibition of the growth of echinococcosis lesions was achieved. and the recovery of liver function provide new treatment pathways.
Patent Information
- Application Number
- CN202411828686.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-12
- Publication Date
- 2025-02-11
AI Technical Summary
Existing treatment methods for echinococcosis, such as albendazole, have low blood concentrations, poor intestinal absorption, and adverse reactions and drug resistance problems caused by long-term use, resulting in unsatisfactory treatment effects.
The GHSR antagonist [D-Lys3]-GHRP-6 is used as a novel therapeutic drug to prepare products for the treatment and/or prevention of echinococcosis by inhibiting GHSR gene expression or reducing its activity.
GHSR antagonist [D-Lys3]-GHRP-6 can effectively inhibit the growth of lesions of Echinococcusia granules, reduce liver damage, restore the effector function of liver T cells, and provide a new way to treat Echinococcusia granules.
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Figure CN119405776A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the application of a [D-Lys3]-GHRP-6 antagonist in the preparation of a medicine for treating echinococcosis granulosus in the field of biomedicine. Background Art
[0002] This section merely provides background information related to the present disclosure and is not necessarily prior art.
[0003] Cystic Echinococcosis (CE) is a chronic parasitic disease caused by the tapeworm Echinococcosis granulosus (E. granulosus), which often parasitizes the human liver. When the inflammatory microenvironment is suppressed, the progression of the disease may be aggravated. CE is a zoonosis that mainly attacks the liver. Adult worms usually parasitize in the small intestine of the final host, such as dogs, and produce infectious eggs. If humans accidentally ingest these eggs, the larvae in the body will hatch, causing liver inflammation, varying degrees of liver damage, and liver fibrosis. CE is widely distributed around the world. Although the mortality rate of the disease is generally low, it may increase significantly if effective prevention and control measures are not taken, so it remains a public health issue that cannot be ignored.
[0004] Currently, surgical resection of the lesion is the main treatment for patients with echinococcosis (CE). However, many patients are already in the advanced stage of the disease when diagnosed. For those with metastasis or multi-organ recurrence, or those who suffer anaphylactic shock due to internal capsule rupture during surgery, drug treatment becomes particularly critical. The first choice treatment drug recommended by the World Health Organization (WHO) is albendazole (ABZ). For CE patients who undergo surgical treatment, ABZ needs to be taken continuously for at least two years after surgery and be reviewed regularly in the next ten years. For those who cannot undergo surgery, they need to rely on ABZ for long-term treatment. However, ABZ has a low concentration in the blood and poor intestinal absorption. Long-term use may cause a series of adverse reactions such as gastrointestinal and liver function, and may lead to drug resistance. These factors limit its application and make the treatment effect unsatisfactory. Therefore, further research and development of effective CE treatment drugs is of great significance for the prevention and control of echinococcosis.
[0005] GHSR (Growth hormone secretagogue receptor, GHSR), also known as Ghrelin receptor, is a G protein-coupled receptor (GPCR) containing 366 amino acids and having typical seven transmembrane domains. It is mainly secreted by the stomach and has multiple physiological functions. It plays an important role in regulating appetite, promoting growth hormone release, protecting the liver and anti-fibrosis. [D-Lys3]-GHRP-6 antagonists have shown their potential application value in the fields of neuroscience, HIV infection research, obesity and metabolism research, and drug development.
[0006] There are currently no reports on the use of GHSR antagonists as targeted drugs for blocking treatment of echinococcosis. Summary of the invention
[0007] Purpose of the invention: The technical problem to be solved by the present invention is to provide an application of a GHSR antagonist in the preparation of a drug for treating echinococcosis granulosus in view of the deficiencies of the prior art.
[0008] In order to solve the above technical problems, the present invention discloses the following technical solutions:
[0009] In a first aspect, the present invention discloses the use of a GHSR antagonist in the preparation of a product for treating and / or preventing echinococcosis granulosus, or the use of a GHSR antagonist in the treatment and / or prevention of echinococcosis granulosus.
[0010] In some embodiments, the GHSR antagonist is [D-Lys3]-GHRP-6.
[0011] In some embodiments, the treatment and / or prevention of Echinococcus granulosus disease is embodied in inhibiting the growth of lesions in animals infected with Echinococcus granulosus or Echinococcus granulosus, and / or reducing liver damage in animals infected with Echinococcus granulosus or Echinococcus granulosus, and / or restoring the effector function of liver T cells in animals infected with Echinococcus granulosus or Echinococcus granulosus.
[0012] In the above applications, the product may be a medicine.
[0013] In a second aspect, the present invention discloses the use of a substance that inhibits GHSR gene expression or knocks out GHSR gene or reduces GHSR content or reduces GHSR activity in the preparation of a product for treating and / or preventing echinococcosis granulosus, or in treating and / or preventing echinococcosis granulosus.
[0014] Wherein, the treatment and / or prevention of echinococcosis is embodied in inhibiting the growth of lesions in animals infected with Echinococcus granulosus or Echinococcus granulosus, and / or alleviating liver damage in animals infected with Echinococcus granulosus or Echinococcus granulosus, and / or restoring the effector function of liver T cells in animals infected with Echinococcus granulosus or Echinococcus granulosus.
[0015] In the above applications, the product may be a medicine.
[0016] In a third aspect, the present invention discloses the use of a GHSR antagonist in preparing a product for inhibiting infection with Echinococcus granulosus or Echinococcus granulosus, or in inhibiting infection with Echinococcus granulosus or Echinococcus granulosus.
[0017] Wherein, the GHSR antagonist is [D-Lys3]-GHRP-6.
[0018] In the above applications, the product may be a medicine or a vaccine.
[0019] In a fourth aspect, the present invention discloses the use of a substance that inhibits GHSR gene expression or knocks out the GHSR gene or reduces the GHSR content or reduces the GHSR activity in the preparation of a product for inhibiting infection with Echinococcus granulosus or Echinococcus granulosus, or in inhibiting infection with Echinococcus granulosus or Echinococcus granulosus.
[0020] In the above applications, the product may be a medicine or a vaccine.
[0021] In the present invention, the alleviation of liver damage can be reflected in the reduction of the content of alanine aminotransferase (ALT) and / or aspartate aminotransferase (AST) in serum.
[0022] In the present invention, the recovery of the effector function of the liver T cells can be reflected in the increase of the levels of factors secreted by T cells, such as INF-γ, IL-2, and IL-6 cytokines, and the decrease of the level of IL-4 cytokine.
[0023] Beneficial effects:
[0024] The present invention provides a new use of the GHSR antagonist, which has therapeutic effects on the growth inhibition of echinococcosis lesions and the recovery of liver function, which means that the GHSR antagonist can be used as a new anti-echinococcosis drug, thereby providing a new approach for the treatment of echinococcosis, and has good clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS
[0025] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more clear.
[0026] Figure 1Schematic diagram of the operation of the GHSR antagonist [D-LYS3]-GHRP-6 in treating the mouse model of echinococcosis granulosus.
[0027] Figure 2 This is an evaluation of the therapeutic effect of the GHSR antagonist [D-LYS3]-GHRP-6 on the mouse model of echinococcosis - liver HE diagram.
[0028] Figure 3 To evaluate the therapeutic effect of the GHSR antagonist [D-LYS3]-GHRP-6 on the mouse model of Echinococcus granulosus - parasitized liver weight.
[0029] Figure 4 Evaluation of liver function in a mouse model of Echinococcus granulosus treated with the GHSR antagonist [D-LYS3]-GHRP-6.
[0030] Figure 5 The levels of cytokine secretion by liver T cells in the mouse model of Echinococcus granulosus treated with the GHSR antagonist [D-LYS3]-GHRP-6 were measured by ELISA.
[0031] Figure 6 The levels of cytokines secreted by liver T cells in a mouse model of Echinococcus granulosus treated with the GHSR antagonist [D-LYS3]-GHRP-6 were detected by qRT-PCR.
[0032] Figure 7 The cytokine secretion capacity of liver T cells in a mouse model of echinococcosis treated with the GHSR antagonist [D-LYS3]-GHRP-6 was examined by flow cytometry.
[0033] Figure 8 The liver proliferation capacity of the mouse model of Echinococcus granulosus treated with the GHSR antagonist [D-LYS3]-GHRP-6 was detected by WB. DETAILED DESCRIPTION
[0034] The present invention can be better understood according to the following examples. However, it is easy for those skilled in the art to understand that the contents described in the examples are only used to illustrate the present invention, and should not and will not limit the present invention described in detail in the claims.
[0035] The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.
[0036] The quantitative tests in the following examples were all repeated three times, and the results were averaged.
[0037] Example Application of GHSR antagonist [D-LYS3]-GHRP-6 as a drug for the treatment of echinococcosis 1 Establishment of an animal model of echinococcosis infected mice
[0038] 1.1 Preparation of Echinococcus granulosus protoscoleces
[0039] The liver infected with Echinococcus granulosus was obtained from a local slaughterhouse. The liver was disinfected and wiped with dry toilet paper. Then, the cystic fluid in the vesicle was aspirated with a 50 mL syringe under sterile conditions, the vesicle was cut open to collect the inner cyst, and the cystic fluid and vesicular tissue were collected with a 50 mL centrifuge tube. The vesicular tissue was cut into pieces, filtered and precipitated with a 100-mesh steel cell sieve, the supernatant was discarded and resuspended with PBS, and the supernatant was discarded again and processed to collect the original PSCs. PSCs were digested with 1% pepsin in saline at 37°C for 30 minutes, and the pH was adjusted to 3.0. The activity of the worm body was calculated by staining with 0.1% methylene blue for 1 minute. The activity was above 95% before it could be used for subsequent model establishment.
[0040] 1.2 Establishment of an animal model of Echinococcus granulosus infection in mouse liver via portal vein
[0041] Adjust the concentration of the isolated protoscoleces of Echinococcus granulosus so that the total system is 200-300 microliters (μL) containing 2000 protoscoleces. Select female BALB / c mice aged 6-8 weeks (Animal Experiment Center of Xinjiang Medical University), inject the protoscoleces of Echinococcus granulosus through needle puncture through the portal vein, and press the puncture point with a dry cotton swab for about 2 minutes (min) until bleeding stops. Place the postoperative mice on a 30℃ electric blanket for recovery until they wake up and return to the mouse cage for normal feeding.
[0042] Treatment with the 2GHSR antagonist [D-LYS3]-GHRP-6 (CAS:136054-22-3) can reduce liver lesions and liver function levels in mice infected with Echinococcus granulosus.
[0043] 2.1 Grouping:
[0044] The experimental groups were model group and treatment group. Female BALB / c mice aged 6 to 8 weeks (18±2g, Animal Experiment Center of Xinjiang Medical University) were used. The model group and treatment group were mice with uniform infection degree after step 1 was completed and after infection with Echinococcus granulosus.
[0045] The experimental groups were model group (2000 protoscoleces were injected into the portal vein) and treatment group (2000 protoscoleces were injected into the portal vein and [D-LYS3]-GHRP-6 was injected intraperitoneally for 12 weeks). There were 5 mice in each group. [D-LYS3]-GHRP-6 was dissolved in DEPC water (0.5 μg / ul). The treatment group was administered with a dose of 200 ul / mouse / day, and intraperitoneal injection was continued for 6 consecutive days per week for a total of 12 weeks (i.e., 12 weeks of treatment). The solvent control group was injected with an equal volume of PBS. The specific operation is shown in Figure 1 .
[0046] 2.2 Evaluation of therapeutic efficacy after 12 weeks of treatment with the GHSR antagonist [D-LYS3]-GHRP-6:
[0047] H&E staining: After embedding, the tissue was sliced onto a slide, dewaxed with xylene, hydrated with gradient ethanol, stained in hematoxylin solution for about 30 seconds, differentiated with hydrochloric acid and ethanol for 1 second, then anti-blued with PBS for 1 minute, stained with eosin for 1 minute, dehydrated again, and sealed after being transparentized with xylene.
[0048] The experimental results are shown in Figure 2 In the Model group, there was inflammatory cell infiltration around the liver cells, including lymphocytes, plasma cells, neutrophils, eosinophils, etc. The liver cells became enlarged and edematous, and eosinophilic degeneration occurred. Some lesions showed lytic necrosis. Long-term verification showed that the infiltrated lesions had ischemic necrosis and cavities. In the [D-LYS3]-GHRP-6 treatment group, the liver cells were arranged relatively neatly, and the inflammatory cell infiltration around the liver cells was reduced, which reduced the pathological damage of the liver, repaired the damaged liver cells, and improved the damaged liver function.
[0049] Liver weight (including lesion weight): The mice were killed, the abdomen was opened to remove the complete liver (including parasitic lesions) of the mice, and the weight of the entire liver was weighed.
[0050] The experimental results are shown in Figure 3 Compared with the Model group, the liver weight (including lesion weight) of the mouse model in the [D-LYS3]-GHRP-6 treatment group was significantly reduced (P<0.05). This result shows that the GHSR antagonist [D-LYS3]-GHRP-6 can inhibit the growth of lesions in the mouse model of echinococcosis and reduce liver damage through intraperitoneal administration.
[0051] 3 Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) content: Take peripheral blood of mice in a 1.5 mL centrifuge tube, let stand at 4°C for 2 hours, centrifuge at 3000 rpm for 15 minutes, separate serum and transfer to a clean centrifuge tube. Use ALT and AST detection kits for detection. The kits were purchased from Nanjing Lapuda Biotechnology Company.
[0052] The experimental results are shown in Figure 4 Compared with the Model group (model group), the levels of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the [D-LYS3]-GHRP-6 treatment group were lower than those in the Model group, and the decrease in AST was more obvious. The obvious decrease in AST and the insignificant decrease in ALT may be related to multiple factors such as the degree of liver damage, drug effects, the stage of liver disease, physiological factors, and lifestyle habits. The results show that the GHSR antagonist [D-LYS3]-GHRP-6 can inhibit the growth of lesions in the mouse model of echinococcosis granulosus and reduce liver damage through intraperitoneal administration.
[0053] 4 Effects of GHSR antagonist [D-LYS3]-GHRP-6 on T cell factors after 12 weeks of treatment:
[0054] 4.1 T cell factor content: Take about 50 mg of mouse liver tissue, add physiological saline and place in a grinding tube for grinding, centrifuge at 8000rpm for 10 minutes, and transfer the supernatant to a clean centrifuge tube. Use IL-2, INF-γ, IL-4, and IL-6 detection kits for detection. The kits were purchased from Nanjing Lapuda Biotechnology Company.
[0055] 4.2T cytokine mRNA level: Take about 50 mg of mouse liver tissue, add 1 ml Trizol and magnetic beads to grind in a grinder, centrifuge at 12000 rpm for 10 min, take the supernatant to a 1.5 ml Ep tube, add 200 uL chloroform, mix well, let stand, and centrifuge again at 12000 rpm for 10 min. The aqueous phase is RNA, add isopropanol of equal proportion, mix well, centrifuge at 12000 rpm for 10 min, discard the supernatant and wash twice with 75% anhydrous ethanol to extract mouse liver tissue RNA. Subsequently, the extracted mRNA was reverse transcribed into cDNA using the HiScript III RTSuperMix for qPCR kit (Vazyme, Nanjing, China). The qRT-PCR reaction was performed using the SYBR Green I detection kit (Vazyme, Nanjing, China), and the changes in the fluorescence signal were monitored using the Bio-Rad real-time PCR system (Bio-Rad, Foster City, CA), where the primers were synthesized by Sangon.
[0056] The experimental results are shown in Figure 5 and Figure 6Compared with the Model group, the levels of T cell secretion factors IL-2, INF-γ, and IL-6 in the [D-LYS3]-GHRP-6 treatment group were increased, and the level of IL-4 was significantly lower than that in the Model group. This result shows that the GHSR antagonist [D-LYS3]-GHRP-6 can regulate the immune response by affecting the secretion of T cell factors through intraperitoneal administration.
[0057] 5. Effect of GHSR antagonist [D-LYS3]-GHRP-6 on the secretion of T cells after 12 weeks of treatment:
[0058] 5.1 Isolation of mouse liver mononuclear cells: Step 2 After 12 weeks of treatment with the GHSR antagonist [D-LYS3]-GHRP-6, liver tissue specimens of mice in the [D-LYS3]-GHRP-6 group and the control group were collected, ground in a plate with PBS, filtered through a steel mesh, and centrifuged at 4°C, 1500 rpm, for 5 min. The precipitate was taken and resuspended with 6 ml of 40% Percoll (Biolegend) solution. The lower layer of cell precipitates was collected by density gradient centrifugation (25°C, 2000 rpm, 30 min), and lysed with red blood cell lysis buffer (Solarbio) at 4°C in the dark for 10 min. Finally, the cells were collected and counted to obtain mouse liver mononuclear cells.
[0059] 5.2 Take 1×10 6 The liver mononuclear cells of mice in the [D-LYS3]-GHRP-6 group and the control group were placed in a 1.5 mL centrifuge tube and cultured in 200 μL of RPMI1640 (Vivacell) containing 10% fetal bovine serum in a 37°C cell culture incubator for 4 hours, and cell stimulants (100-fold dilution, Biolegend) were added at the same time. The cells were collected and washed with PBS, blocked with CD16 / 32 antibody (Biolegend), and then treated with a fixation and permeabilization kit (Biolegend), and the intracellular cytokines IL-4 and IL-17 were stained and labeled, and detected by flow cytometry.
[0060] The experimental results are shown in Figure 7 Compared with the model group, the ability of CD4+T cells in the liver of the mouse model treated with the GHSR antagonist [D-LYS3]-GHRP-6 to secrete molecules IL-4 and IL-17 was reduced. This result shows that the GHSR antagonist [D-LYS3]-GHRP-6 can restore the effector function of liver T cells through intraperitoneal treatment.
[0061] 6 Protein level detection of proliferation factors after 12 weeks of treatment with GHSR antagonist [D-LYS3]-GHRP-6:
[0062] About 50 mg of mouse liver tissue was taken, RIPA: phosphatase inhibitor: protease inhibitor = 100: 1: 1 was added to the magnetic beads and ground in a grinder. After standing at 4°C for 30 min, the supernatant was centrifuged at 12000 rpm for 10 min and the supernatant was taken into a 1.5 ml Ep tube. The concentration was determined using BCA protein concentration (Solarbio). Proteins were separated using SDS-PAGE gel (Biotides, Beijing, China) and transferred to PVDF electrotransfer membrane (Sigma-Aldrich, Shanghai, China). The membrane was treated in a blocking solution containing 5% skim milk powder and incubated with the primary antibody solution at 4°C overnight. After washing the next day, it was combined with HRP-labeled secondary antibody (multi-rAb HRP-goatanti-rabbit, 1:5000, RGAR001, Proteintech, Wuhan, China). Finally, the enhanced ECL kit (Biosharp, Hefei, China) was used for color development to detect the target protein.
[0063] The experimental results are shown in Figure 8 Compared with the model group, the GHSR antagonist [D-LYS3]-GHRP-6 treated mice had reduced liver Cyclin D1 and Cyclin E1 activity. This may indicate that the GHSR antagonist has the ability to inhibit cell proliferation, regulate cell cycle progression, and affect metabolism and energy balance.
[0064] The above results show that the GHSR antagonist [D-LYS3]-GHRP-6 treatment can effectively inhibit the lesion growth of animals infected with Echinococcus granulosus or Echinococcus granulosus, alleviate liver damage, reduce the ability of liver T cells to secrete effector molecules, and reduce the proliferation ability relative to the protein level. This shows that the GHSR antagonist [D-LYS3]-GHRP-6 has a therapeutic effect on Echinococcus granulosus disease.
[0065] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.
Claims
1. Use of GHSR antagonists in the preparation of products for treating and / or preventing echinococcosis.
2. The use according to claim 1, characterized in that: The GHSR antagonist is [D-Lys3]-GHRP-6.
3. The use according to claim 1, characterized in that: The treatment and / or prevention of echinococcosis is embodied in inhibiting the growth of lesions in animals infected with Echinococcus granulosus or Echinococcus granulosus, and / or alleviating liver damage in animals infected with Echinococcus granulosus or Echinococcus granulosus, and / or restoring the effector function of liver T cells in animals infected with Echinococcus granulosus or Echinococcus granulosus.
4. Use of a substance that inhibits GHSR gene expression or knocks out GHSR gene or reduces GHSR content or reduces GHSR activity in the preparation of a product for treating and / or preventing echinococcosis granulosus.
5. The use according to claim 4, characterized in that: The treatment and / or prevention of echinococcosis is embodied in inhibiting the growth of lesions in animals infected with Echinococcus granulosus or Echinococcus granulosus, and / or alleviating liver damage in animals infected with Echinococcus granulosus or Echinococcus granulosus, and / or restoring the effector function of liver T cells in animals infected with Echinococcus granulosus or Echinococcus granulosus.
6. The use according to claim 1 or 4, characterized in that: The products include pharmaceutical products.
7. Use of GHSR antagonists in the preparation of products for inhibiting infection by Echinococcus granulosus or Echinococcus granulosus.
8. The use according to claim 7, characterized in that: The GHSR antagonist is [D-Lys3]-GHRP-6.
9. Use of a substance that inhibits GHSR gene expression or knocks out GHSR gene or reduces GHSR content or reduces GHSR activity in the preparation of a product for inhibiting infection by Echinococcus granulosus or Echinococcus granulosus.
10. The use according to claim 7 or 9, characterized in that: The product includes a medicine or a vaccine.