Application of TLR4 protein in treatment of perimenopausal depression

Abstract

The invention discloses application of TLR4 protein in treatment of perimenopausal depression, and belongs to the technical field of treatment of perimenopausal depression. The invention reveals the important effect of the TLR4 protein in the perimenopausal depression for the first time, provides a new potential therapeutic target for the perimenopausal depression, and provides a new idea for the treatment of the perimenopausal depression; experiments in the invention show that the TLR4 inhibitor can effectively improve depression-like behaviors caused by ovarian function decline or disappearance, can also relieve neuron damage, has a good protection effect on neurons, and has important application prospects.

Description

Technical Field

[0001] The present invention relates to the technical field of perimenopausal depression treatment, and in particular to application of TLR4 protein in perimenopausal depression treatment. Background Art

[0002] Perimenopausal depression (PMDD) is a common disease that affects women's mental health. Especially in the context of decreased ovarian function and changes in hormone levels, many women experience mood swings, depression, and anxiety symptoms during the perimenopausal stage. Current drugs for treating perimenopausal depression include antidepressants and hormone replacement therapy, but these treatments are not completely effective and may be accompanied by side effects. Therefore, it is particularly important to find new targets and treatments.

[0003] TLR4 (Toll-like receptor 4) is a protein located on the cell surface and an important member of the pattern recognition receptor (PRRs) family. It plays a key role in the innate immune system and can recognize pathogen-associated molecular patterns (PAMPs) such as lipopolysaccharide (LPS). It is widely involved in the pathological mechanisms of infectious diseases, chronic inflammation, metabolic disorders and cancer, and is an important target for immune regulation and targeted therapy. Summary of the invention

[0004] The purpose of the present invention is to provide the use of TLR4 protein in the treatment of perimenopausal depression, so as to provide a new potential therapeutic target for the treatment of perimenopausal depression and provide a new treatment idea for perimenopausal depression.

[0005] To achieve the above object, the present invention provides the use of TLR4 protein in the treatment of perimenopausal depression, wherein the TLR4 protein is a target for the treatment of perimenopausal depression.

[0006] Preferably, the expression of the TLR4 protein is inhibited to improve perimenopausal depression.

[0007] Application of a TLR4 protein inhibitor in the preparation of a drug for treating perimenopausal depression, wherein the drug uses the TLR4 protein inhibitor as an active ingredient.

[0008] Application of a TLR4 protein inhibitor in the preparation of a neuroprotective drug, wherein the drug uses the TLR4 protein inhibitor as an active ingredient.

[0009] Therefore, the application of TLR4 protein provided by the present invention in the treatment of perimenopausal depression has the following specific technical effects:

[0010] (1) The present invention reveals for the first time the important role of TLR4 protein in perimenopausal depression, provides a new potential therapeutic target for perimenopausal depression, and provides a new idea for the treatment of perimenopausal depression;

[0011] (2) The experiments in the present invention show that TLR4 inhibitors can effectively improve depressive-like behaviors caused by decreased or absent ovarian function, and can also reduce neuronal damage and have a good protective effect on neurons, thus having important application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for use in the description of the embodiments of the present invention will be briefly introduced below. Obviously, the accompanying drawings in the following description are only some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying creative labor.

[0013] Figure 1 It is the FST result in Example 4 of the present invention; wherein part a is the statistical result of the immobility time of rats within 5 minutes; part b is the statistical result of the latency before the onset of the immobility time; * / # indicates P<0.05, ** indicates P<0.001, and ns indicates no significant difference;

[0014] Figure 2 is the SPT result in Example 4 of the present invention; wherein ** indicates P<0.001, ns indicates no significant difference, and # indicates P<0.05;

[0015] Figure 3 It is the result of protein immunoblotting analysis in Example 5 of the present invention; wherein part a is the result of TLR4 / β-actin, part b is the result of p-p65 / p65, part c is the result of p65 / β-actin, part d is the result of MyD88 / β-actin, part e is the result of TNF-α / β-actin, part f is the result of IL-6 / β-actin, and part g is the result of IL-1β / β-actin; * / # indicates P<0.05, ** / ## indicates P<0.001, *** / ### indicates P<0.0001, **** / #### indicates P<0.00001, and ns indicates no significant difference;

[0016] Figure 4 is the co-staining result of TLR4 and NEUN in Example 6 of the present invention; wherein ns indicates no significant difference, *** indicates P<0.0001, and **** indicates P<0.00001;

[0017] Figure 5 This is the result of TUNEL and NEUN co-staining in Example 6 of the present invention; ns indicates no significant difference, and ** / ## indicates P<0.001. DETAILED DESCRIPTION

[0018] The technical solution of the present invention is further described below through the accompanying drawings and embodiments.

[0019] In order to make the purpose, technical scheme and advantages of the present application clearer, more thorough and more complete, the technical scheme of the present invention is clearly and completely described below through the accompanying drawings and examples. The following detailed descriptions are all descriptions of the embodiments, and are intended to provide further detailed descriptions of the present invention. Unless otherwise specified, all technical terms used in the present invention have the same meaning as those generally understood by those skilled in the art to which the present application belongs.

[0020] The instruments, equipment and reagents used in the examples were obtained commercially; the adult female SD rats used were clean grade, aged 8 to 10 weeks, weighing 250 to 280 g, and were kept in 45×25×15 cm polycarbonate cages, 2 to 3 rats per cage. The appropriate temperature (22 to 24°C), humidity (50 to 70%) and regular light-dark cycle (lights on at 7 a.m. and off at 7 p.m.) were maintained, and rats were free to get food and water in the cages. The experiment was started after a week of adaptation. All animal studies and experiments in the examples of the present invention were approved by the Ethics Committee for the Use and Care of Experimental Animals of Ningbo University.

[0021] Embodiment 1

[0022] The specific steps for constructing a perimenopausal depression model are as follows:

[0023] S11. Select 16 healthy and active clean-grade female SD rats weighing 250-280g and perform ovariectomy: weigh the rats and inject 0.1mL / kg of Zoletil 50 intraperitoneally for anesthesia. Shave the hair on the abdomen with a razor, clean and disinfect with povidone iodine, and use a sterile scalpel to make a 2cm incision in the middle of the abdomen. The bladder can be easily seen. Find the Y-shaped uterus behind the bladder, find the right fallopian tube ovary along the right uterus, clamp and remove the right fallopian tube ovary, and tie the stump with surgical silk. Remove the left fallopian tube and ovary in the same way, and suture the abdominal incision.

[0024] S12. After surgery, the rats were placed in a clean constant temperature cage (45×25×15cm, temperature 22-24°C, humidity 50-70%). The estrus cycle of rats is 4 days, including proestrus, estrus, metestrus and estrus. After surgery, the rats were taken out of the cage at 8 am every day, the vagina was exposed, 80 μL of normal saline was drawn with a pipette, gently inserted into the rat's vagina, and then blown two or three times. The resulting liquid was placed on a glass slide, naturally air-dried, and then quickly Gram-stained, and then sealed with neutral gum to prepare a vaginal smear. The cell types in the smear were observed under a microscope to evaluate the estrus cycle of the rats. Continuous smear observations showed that the rats were successfully castrated, the estrus cycle became irregular, and the rats were in estrus after 7 days, indicating that the perimenopausal depression model was successfully constructed.

[0025] Embodiment 2

[0026] The sham operation group of rats was constructed, and the specific steps were as follows:

[0027] S21. Eight healthy and active clean-grade female SD rats weighing 250-280 g were selected for sham surgery: the rats were weighed and anesthetized with an intraperitoneal injection of 0.1 mL / kg of Zoletil 50, and the hair on the abdomen was shaved with a razor. After cleaning and disinfection with povidone iodine, a 2 cm incision was made in the middle of the abdomen with a sterile scalpel. The bladder was easily visible, and the Y-shaped uterus behind the bladder was found. The right fallopian tube and the fat tissue around the ovary were searched along the right uterus, clamped and removed, and then the stump was tied with surgical silk. The same method was used to remove the same size of fat tissue around the left fallopian tube and ovary, and the abdominal incision was sutured.

[0028] S22. After surgery, the rats were placed in a clean constant temperature cage (45×25×15 cm, temperature 22-24° C., humidity 50-70%) for routine culture. The sham operation group rats were obtained.

[0029] Embodiment 3

[0030] The TLR4 inhibitor was used to treat the perimenopausal depression model mice constructed in Example 1, and the specific steps were as follows:

[0031] Take 8 healthy and active clean female SD rats weighing 250-280g, and record them as the control group (CTL). Take 8 sham-operated rats constructed in Example 2, and record them as the sham-operated group (SHAM). Take 8 perimenopausal depression model rats constructed in Example 1, and record them as the ovariectomized group (OVX). Take 8 perimenopausal depression model rats constructed in Example 1, and inject 10mg / kg dose of ibudilast (a TLR4 inhibitor) intraperitoneally 2 hours before all behavioral tests, and record them as the ibudilast group (IBU). The control group, sham-operated group and ovariectomized group rats were injected with 10mg / kg dose of normal saline 2 hours before all behavioral tests.

[0032] Embodiment 4

[0033] The rats in each group in Example 3 were subjected to behavioral tests, using a sugar water preference test and a forced swimming test to assess depressive behavior. Before all behavioral assessments, the rats in each group were confirmed to be in estrus by vaginal smear analysis, and then the following tests were performed:

[0034] (1) Forced swim test (FST)

[0035] FST is an indicator of despair-related behavior in rats, which is related to depressive states. Rats were placed in a cylinder (50 cm high, 20 cm in diameter, 30 cm deep) filled with water at a temperature of 23-25°C. A camera recorded the immobility time of the rats within five minutes, as well as the latency before the onset of the immobility period. After each test, the rats were dried and returned to the cage, the water in the cylinder was poured out, and the cylinder was scrubbed before the subsequent test of the rats.

[0036] (2) Sucrose preference test (SPT)

[0037] SPT was used to evaluate whether rats had anhedonia. The test consisted of two phases: an adaptation phase and a test phase. Routine feeding was performed from day 1 to day 43. The adaptation phase began on day 44, during which bottles containing 1% sucrose solution were placed on both sides of the cage. Then, on day 45, a bottle of pure water was placed to replace a bottle of sucrose solution (the positions of the two water bottles were swapped after 12 h to eliminate the possible influence of position preference). On day 46, water was deprived for 24 hours. The test phase occurred on day 47. Before the test, the bottles (one containing 1% sucrose solution and the other containing pure water) were weighed and placed on both sides of the cage. The positions were swapped after half an hour and the bottles were taken out and weighed after one hour. The consumption levels of sucrose solution and pure water were carefully recorded to calculate the sugar water preference index (%) = [sucrose solution consumption / (sucrose solution consumption + pure water consumption)] × 100%.

[0038] Embodiment 5

[0039] Western blotting analysis was performed on the brain tissues of rats in each group in Example 3, as follows:

[0040] The rats after the behavioral test in Example 4 were anesthetized and then decapitated. Brain tissue was extracted on ice (4 rats per group). The specific brain area was quickly selected using a mold, and the rat mPF C tissue was extracted and stored in the corresponding labeled EP tube. The protein concentration was determined by the BCA method (Beyotime, Institute of biotechnology). The total protein was then separated by 10% SDS-PAGE (a total of 40 μg was obtained), and then transferred to a polyvinylidene fluoride membrane (Bio-Rad, Laboratories, Inc., USA), and the membrane was blocked with 3% BSA-TBST for 1 hour, and incubated at 4°C with rabbit monoclonal antibodies (anti-tlr4 (1:1000; #PA5-23124; Invitrogen), Phospho-NF-κB p65 (1:1000; #MA5-15160; Invitrogen), NF-κB p65 (1:1000; #8242; CST), IL-1β (1:1000; #Ab254360; Abcam), IL-6 (1:1000; #Ab9324; Abcam), TNF-α (1:1000; #Ab183218; Abcam), and mouse monoclonal antibodies MyD88 (1:5000; #sc-74532; Santacruz), caspase3 (1:1000; #9662; CST), and β-actin (1:25000; #A3854; Sigma-Aldrich) were incubated for 1 h at room temperature after thorough washing. After washing three times with TBST, chemiluminescent developer was added and incubated in a biochemical imaging system. Finally, protein quantification was performed using image processing and analysis software (ImageJ, USA), and relative density values ​​were calculated using β-actin as an internal control.

[0041] Embodiment 6

[0042] Immunofluorescence analysis was performed on the brain tissues of the rats in each group in Example 3, as follows:

[0043] The rats after the behavior test in the fourth embodiment were anesthetized, then fixed on the operating board, cut along the ribs with scissors, and fully exposed the chest cavity. A small incision was made gently on the left side of the apex of the heart, and the apex of the heart and the aortic valve were penetrated with a perfusion needle to reach the aorta. The heart and the perfusion needle were then fixed with hemostatic forceps to open the liquid to the maximum extent. The right auricle was cut with scissors to make the blood flow out, and 200mL0.9% sodium chloride injection was poured in first until the liver and lungs turned white. This process is to discharge the blood in the brain, and then 200mL4% paraformaldehyde solution was immediately poured into the perfusion bottle that was about to be emptied, and the first 100mL continued to be perfused at a high flow rate, and after 100mL, the speed was slowed down. After perfusion, the brain tissue was cut off with a guillotine, and the brain tissue was immersed in 4% paraformaldehyde and numbered. The brain tissues were embedded in paraffin and cut into 5 μm sections, followed by dewaxing and antigen retrieval, and then the sections blocked with 3% bovine serum albumin were co-stained with anti-tlr4 (1:250; #PA5-23124; Invitroge), NEUN (1:400; #94403; CST), IBA1 (1:100; #Ab283319; Abcam), GFAP (1:500; #3670; CST), PV (1:100; #P3088; Sigma-Aldrich), and CAMKII (1:500; #Ab22609; Abcam). The antigen-binding primary antibodies used were Alexa FluorTM 488 goat anti-mouse IgG (H+L) (1:500; #A11001; Invitrogen) and Alexa FluorTM 594 goat anti-rabbit IgG (H+L) (1:500; #A11012; Non-Neutron) secondary antibodies. All sections were examined under a fluorescence microscope (OLYMPUSBX53, Tokyo, Japan). Under ultraviolet light excitation, DAPI staining showed that the cell nucleus was blue, and the red or green fluorescence corresponding to the labeled antibody indicated positive expression. TUNEL staining was performed using a one-step TUNEL apoptosis assay kit (Beyotime #C1090) combined with an anti-neuun primary antibody.

[0044] Results Analysis

[0045] All statistical data were analyzed using GraphPad Prism 7.0 software (San Diego, CA, USA) and expressed as mean ± standard error. The differences in means between groups were evaluated using one-way analysis of variance (ANOVA), Tukey's multiple comparisons or unpaired t-test, and P < 0.05 was considered significant.

[0046] (1) The FST results in Example 4 are as follows Figure 1 As shown, compared with the CTL group and the SHAM group, the immobility time in the OVX group was significantly prolonged, and the latency to reach the immobility time was decreased, which was improved after the administration of the TLR4 inhibitor (IBU group).

[0047] SPT results are as follows Figure 2 As shown, the sucrose preference index of rats in the OVX group was significantly lower than that in the CTL group and the SHAM group, and was improved after administration of the TLR4 inhibitor (IBU group).

[0048] The results showed that ovariectomized rats showed obvious depressive-like behavior, and TLR4 inhibitors could improve the depressive-like behavior.

[0049] (2) The results of the protein immunoblotting analysis in Example 5 are as follows Figure 3 As shown in the results, TLR4, MyD88, NF-κB and proinflammatory cytokines (TNF-α, IL-1β and IL-6) were significantly increased in the prefrontal cortex of ovariectomized rats, which was improved after administration of TLR4 inhibitors. The increased TLR4 was mainly expressed on neurons and induced neuronal apoptosis.

[0050] (4) The immunofluorescence analysis results in Example 6, TLR4 and NEUN co-staining results are as follows Figure 4 As shown, the expression of TLR4 on neurons in the OVX group was increased compared with the CTL group and the SHAM group, and was decreased after administration of the TLR4 inhibitor (IBU group).

[0051] The results of TUNEL and NEUN co-staining are as follows Figure 5 As shown, co-staining of TUNEL and neurons also shows that neuronal apoptosis in ovariectomized rats increased, which was improved after administration of TLR4 inhibitors (IBU group). (TUNEL-NEUN is the number of co-staining)

[0052] Therefore, the present invention reveals for the first time the important role of TLR4 protein in perimenopausal depression, provides a new potential therapeutic target for perimenopausal depression, and provides a new idea for the treatment of perimenopausal depression; the experiments in the present invention show that TLR4 inhibitors can effectively improve depressive-like behaviors caused by decreased or disappeared ovarian function, and can also reduce neuronal damage and have a good protective effect on neurons, which has important application prospects.

[0053] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solution of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solution to deviate from the spirit and scope of the technical solution of the present invention.

Claims

1. The use of TLR4 protein in the treatment of perimenopausal depression, characterized in that: The TLR4 protein is a target for the treatment of perimenopausal depression.

2. The use of TLR4 protein in the treatment of perimenopausal depression according to claim 1, characterized in that: Inhibiting the expression of the TLR4 protein improves perimenopausal depression.

3. The use of a TLR4 protein inhibitor in the preparation of a drug for treating perimenopausal depression, characterized in that: The drug uses a TLR4 protein inhibitor as an effective ingredient.

4. The use of a TLR4 protein inhibitor in the preparation of a neuroprotective drug, characterized in that: The drug uses a TLR4 protein inhibitor as an effective ingredient.

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