阅读说明：本技术 一种褪黑素在制备治疗自身免疫性前列腺炎药物中的应用、治疗自身免疫性前列腺炎的药物 (Application of melatonin in preparation of medicine for treating autoimmune prostatitis and medicine for treating autoimmune prostatitis ) 是由 陈晶 梁朝朝 张力 金宗兰 张蒙 张礼刚 于 2021-02-18 设计创作，主要内容包括：本发明提供了一种褪黑素在制备治疗自身免疫性前列腺炎药物中的应用、治疗自身免疫性前列腺炎的药物,属于药物应用技术领域。本发明通过增强Sirt1通路以及抑制NLRP3炎症小体从而减轻前列腺组织的炎性改变、疼痛反应及外周血中致炎性细胞因子的含量。本发明以自身免疫性前列腺炎鼠模型为试验对象,研究发现褪黑素通过增强Sirt1通路、抑制NLRP3炎症小体从而明显减轻前列腺组织的炎性改变、疼痛反应及外周血中致炎性细胞因子的含量,即褪黑素通过Sirt1依赖性抑制NLRP3炎性小体的表达从而减轻前列腺炎症,褪黑素对于前列腺炎的治疗是可行有效的。(The invention provides application of melatonin in preparation of a medicine for treating autoimmune prostatitis and a medicine for treating autoimmune prostatitis, and belongs to the technical field of medicine application. The present invention alleviates the inflammatory changes in prostate tissue, pain response and proinflammatory cytokine levels in peripheral blood by enhancing the Sirt1 pathway and inhibiting NLRP3 inflammasome. The invention takes an autoimmune prostatitis mouse model as a test object, researches show that melatonin obviously reduces inflammatory change, pain reaction and content of proinflammatory cytokines in peripheral blood by enhancing a Sirt1 passage and inhibiting NLRP3 inflammasome, namely, melatonin reduces prostatitis by inhibiting expression of NLRP3 inflammasome in a Sirt1 dependence mode, and is feasible and effective for treating prostatitis.)

1. An application of melatonin in preparing medicine for treating autoimmune prostatitis is disclosed.

2. The use of claim 1, wherein the autoimmune prostatitis is induced by prostate antigen.

3. The use as claimed in claim 1, wherein the melatonin is used in the form of an injection solution, and the preparation method of the injection solution of melatonin comprises: dissolving melatonin in dimethyl sulfoxide, and mixing the obtained solution with physiological saline to obtain melatonin injection.

4. The use according to claim 3, wherein the concentration of the melatonin injection is 3-8 mg/mL.

5. A medicine for treating autoimmune prostatitis is characterized in that the active ingredient of the medicine comprises melatonin.

6. The pharmaceutical composition of claim 5, wherein the pharmaceutical composition is in the form of an injection.

Technical Field

The invention relates to the technical field of medicine application, in particular to application of melatonin in preparing a medicine for treating autoimmune prostatitis and a medicine for treating the autoimmune prostatitis.

Background

Chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS), known as prostatitis class III, is a common urological condition in men under 50 years of age, accounting for 90% of all cases of prostatitis, with an incidence of about 8.4%. The most common symptoms of CP/CPPS mainly include chronic pelvic pain occurring in the pelvis, perineum, scrotum and testis. Because the etiology of CP/CPPS is still unclear, current CP/CPPS therapies are unsatisfactory for both patients and physicians. Therefore, intensive research on the etiology of CP/CPPS is particularly important to explore effective therapeutic approaches for CP/CPPS.

NLRP3, caspase-1 and apoptosis-related speckle-like protein (ASC) constitute NLRP3 inflammatory corpuscle and have the function of promoting the maturation of proinflammatory cytokine interleukin 1 beta (IL-1 beta). An increasing number of studies have demonstrated that NLRP3 inflammasome is associated with a range of chronic inflammatory diseases. Currently, many studies have shown that NLRP3 inflammasome and IL-1. beta. are involved in the development and progression of CP/CPPS. Therefore, NLRP3 inflammasome and IL-1. beta. may be one of the targets for treatment of CP/CPPS.

Melatonin (also known as melatonin) is a neuroendocrine hormone that is secreted primarily by the pineal gland in a circadian manner. It has a wide range of biological properties, such as anti-inflammatory, antioxidant, immunomodulating etc. Previous studies have shown that melatonin can exert its anti-inflammatory effects by inhibiting NLRP3 inflammatory bodies in a variety of inflammatory diseases. Sirtuin regulator 1(Sirt1) is a nicotinamide adenine dinucleotide (NAD +) dependent histone deacetylase and studies have shown Sirt1 to be an important regulator of anti-aging, anti-stress and anti-inflammatory properties. Many studies have shown that Sirt1 can exert anti-inflammatory effects by inhibiting NLRP3 inflammasome and IL-1 β. In addition, research reports that Sirt1 expression is reduced in CP/CPPS, and drugs such as resveratrol can inhibit abnormal inflammatory reaction in CP/CPPS by activating Sirt 1. However, there is currently no study on whether melatonin exerts its protective effect on CP/CPPS by activating Sirt 1.

Disclosure of Invention

The invention aims to provide an application of melatonin in preparing a medicine for treating autoimmune prostatitis, and the medicine for treating the autoimmune prostatitis, wherein the melatonin can reduce prostatitis through Sirt 1-dependent inhibition of the expression of NLRP3 inflammasome.

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

the invention provides an application of melatonin in preparing a medicine for treating autoimmune prostatitis.

Preferably, the autoimmune prostatitis is induced by prostate antigen.

Preferably, the melatonin is used in the form of an injection, and the preparation method of the injection of melatonin comprises the following steps: dissolving melatonin in dimethyl sulfoxide, and mixing the obtained solution with physiological saline to obtain melatonin injection.

Preferably, the concentration of the melatonin injection is 3-8 mg/mL.

The invention provides a medicament for treating autoimmune prostatitis, and the active ingredient of the medicament comprises melatonin.

Preferably, the pharmaceutical composition is in the form of injection.

The invention provides an application of melatonin in preparing a medicine for treating autoimmune prostatitis. The invention takes an autoimmune prostatitis mouse model as a test object, researches show that melatonin obviously reduces inflammatory change, pain reaction and content of proinflammatory cytokines in peripheral blood by enhancing a Sirt1 passage and inhibiting NLRP3 inflammasome, namely, melatonin reduces prostatitis by inhibiting expression of NLRP3 inflammasome in a Sirt1 dependence mode, and is feasible and effective for treating prostatitis.

Drawings

FIG. 1 is a graph showing a representative representation of histological staining of prostate gland of mice treated in example 1 and comparative examples 1 to 2, a comparison of pathological change scores, a difference graph of test responses of mice to pelvic pain, and effects of expression levels of IFN-. gamma. (a), IL-17(b), and IL-1. beta. (c) inflammatory cytokines in serum of mice;

FIG. 2 is a graph of immunohistochemical analysis of prostate tissue of mice treated in example 1 and comparative examples 1-2;

FIG. 3 is a Western blot analysis of treated mice of example 1 and comparative examples 1-2 and a plot of the relative densities of NLRP3, ASC, caspase-1, IL-1 β, and Sirt1 bands versus the corresponding β -actin bands;

FIG. 4 is a graph showing a representative representation of histological staining of mouse prostate, a comparison of pathological change scores of mouse prostate, a graph showing a difference in response to a pelvic pain test in mice, and expression levels of IFN-. gamma. (a), IL-17(b), and IL-1. beta. (c) inflammatory cytokines in mouse serum after treatment in examples 1-2 and comparative examples 1-2;

FIG. 5 is a graph of immunohistochemical analysis of prostate tissue of mice treated in examples 1-2 and comparative examples 1-2;

FIG. 6 is a graph of Western blot analysis results and the relative density of NLRP3, ASC, caspase-1, IL-1. beta. and Sirt1 bands versus the corresponding beta-actin bands for the treated mice of examples 1-2 and comparative examples 1-2.

Detailed Description

The invention provides an application of melatonin in preparing a medicine for treating autoimmune prostatitis.

In the present invention, the autoimmune prostatitis is preferably induced by prostate antigen. The source of the prostate antigen is not particularly limited in the present invention, and commercially available products well known in the art are acceptable.

The source of melatonin in the present invention is not particularly limited, and commercially available products known in the art may be used. In the present invention, the melatonin is used in the form of an injection, and the preparation method of the injection of melatonin comprises: dissolving melatonin in dimethyl sulfoxide, and mixing the obtained solution with physiological saline to obtain melatonin injection.

In the invention, the mass concentration of the solution obtained by dissolving melatonin in dimethyl sulfoxide is preferably 0.1-0.2%; the concentration of the physiological saline is not particularly limited in the present invention, and commercially available physiological saline known in the art may be used. The process of mixing the obtained solution with the physiological saline is not particularly limited in the present invention, and the materials can be uniformly mixed according to a process well known in the art.

In the invention, the concentration of melatonin in the melatonin injection is preferably 3-8 mg/mL, and more preferably 4-6 mg/mL. In the invention, the melatonin injection is prepared for use at present and is stored in a dark place. In the present invention, the physiological saline is preferably used in an amount that allows the melatonin injection to be obtained within the above concentration range.

The invention provides a medicament for treating autoimmune prostatitis, and the active ingredient of the medicament comprises melatonin.

In the present invention, the dosage form of the pharmaceutical composition is preferably an injection.

The invention takes an autoimmune prostatitis mouse model as a test object, and researches show that melatonin obviously reduces inflammatory change, pain response and content of proinflammatory cytokines in peripheral blood of prostate tissues by enhancing a Sirt1 passage and inhibiting NLRP3 inflammasome, namely melatonin reduces prostatitis by inhibiting expression of NLRP3 inflammasome dependently through Sirt 1.

The method for researching the action mechanism of the melatonin on the autoimmune prostatitis by taking the autoimmune prostatitis mouse model as a test object preferably comprises the following steps:

establishing an autoimmune prostatitis mouse model EAP, and preparing melatonin injection;

injecting melatonin injection into the abdominal cavity of EAP mice every day from the 28 th day of subcutaneous immunization until the mice are sacrificed at the 42 th day;

the degree of inflammation was assessed by histopathology, EAP mouse plantar pain, peripheral blood ELISA assay, followed by western immunoblot and immunohistochemical assay for expression of nod (nuclear binding oligomerization domain), receptor-like family 3(NLRP3), apoptosis-associated plaque spotting protein (ASC), caspase 1(caspase-1), interleukin-1 (interleukin-1 β, IL-1 β), and silencing information regulator 2homolog 1), Sirt1, in EAP mouse prostate tissue.

The invention establishes an autoimmune prostatitis mouse model EAP, and prepares melatonin injection. In the invention, the method for establishing the autoimmune prostatitis mouse model EAP is preferably as follows: a non-obese diabetic mouse NOD is injected subcutaneously with a mixture of prostate antigen and complete Freund's adjuvant on days 0 and 28, respectively, to form an autoimmune prostatitis mouse model EAP. In the present invention, the volume ratio of the prostate antigen to the complete Freund's adjuvant in the mixture of the prostate antigen and the complete Freund's adjuvant is preferably 1: 1. The source of the prostate antigen and Freund's complete adjuvant is not particularly limited in the present invention, and any commercially available product known in the art may be used.

In the present invention, the preparation method of the melatonin injection preferably comprises: dissolving melatonin in dimethyl sulfoxide, and mixing the obtained solution with physiological saline to obtain melatonin injection.

In the invention, the mass concentration of the solution obtained by dissolving melatonin in dimethyl sulfoxide is preferably 0.1-0.2%; the concentration of the physiological saline is not particularly limited in the present invention, and commercially available physiological saline known in the art may be used. The process of mixing the obtained solution with the physiological saline is not particularly limited in the present invention, and the materials can be uniformly mixed according to a process well known in the art.

In the invention, the concentration of melatonin in the melatonin injection is preferably 3-8 mg/mL, and more preferably 4-6 mg/mL. In the invention, the melatonin injection is prepared for use at present and is stored in a dark place. In the present invention, the physiological saline is preferably used in an amount that allows the melatonin injection to be obtained within the above concentration range.

After obtaining the EAP model of the autoimmune prostatitis mouse, the invention injects melatonin injection into the abdominal cavity of the EAP mouse every day from the 28 th day of subcutaneous immunization until the mouse is sacrificed at the 42 th day.

In the invention, the total injection amount of the melatonin injection injected into the abdominal cavity of the EAP rat every day is preferably 30-80 mg/kg, more preferably 40-60 mg/kg, and even more preferably 45-50 mg/kg, and the volume of the melatonin injection injected each time is preferably 0.2 mL. The injection time is not specifically limited, and the injection time can be adjusted according to actual requirements; in the embodiment of the invention, the injection is specifically performed at 8-9 am every day.

After the mice are sacrificed, the invention evaluates the inflammation degree according to histopathology, the pain detection of the sole of the EAP mouse and the ELISA detection of peripheral blood, and then detects the expression conditions of NLRP3, ASC, caspase-1, IL-1 beta and Sirt1 in the prostate tissue of the EAP mouse according to Western blotting and immunohistochemistry.

The invention has no special limitation on the specific processes of detecting, evaluating and verifying the degree and detecting the expression conditions of NLRP3, ASC, caspase-1, IL-1 beta and Sirt1 in the prostate tissue of an EAP mouse, and the detection is carried out according to the processes well known in the art.

The influence of melatonin on the prostate verification degree of an EAP mouse model is researched by injecting melatonin injection into the EAP mouse.

In the present invention, the method of application preferably further comprises: preparing Sirt1 inhibitor-EX 527 injection, injecting Sirt1 inhibitor-EX 527 injection into the abdominal cavity of the EAP mice every other day from the day before the melatonin injection is injected into the abdominal cavity of the EAP mice, and detecting the degree of the prostate tissue inflammation of the EAP mice.

In the present invention, the preparation method of the Sirt1 inhibitor-EX 527 injection preferably comprises the following steps: EX527 was dissolved in dimethyl sulfoxide, and the resulting solution was mixed with physiological saline to give a Sirt1 inhibitor-EX 527 injection.

The source of EX527 is not particularly limited in the present invention, and commercially available products known in the art may be used. In the invention, the mass concentration of the solution obtained by dissolving EX527 in dimethyl sulfoxide is preferably 0.1-0.2%; the concentration of the physiological saline is not particularly limited in the present invention, and commercially available physiological saline known in the art may be used. The process of mixing the obtained solution with the physiological saline is not particularly limited in the present invention, and the materials can be uniformly mixed according to a process well known in the art.

In the invention, the concentration of the Sirt1 inhibitor-EX 527 in the Sirt1 inhibitor-EX 527 injection is preferably 0.5-1 mg/mL, and more preferably 0.6-0.8 mg/mL. In the present invention, the Sirt1 inhibitor-EX 527 injection is ready for use. In the present invention, the physiological saline is preferably used in an amount that allows the Sirt1 inhibitor-EX 527 injection to be obtained in the above-described concentration range.

In the invention, the total injection amount (namely the total injection amount per day) of the Sirt1 inhibitor-EX 527 injection injected into the abdominal cavity of the EAP rat every other day is preferably 5-10 mg/kg, more preferably 6-9 mg/kg, even more preferably 7-8 mg/kg, and the volume of the Sirt1 inhibitor-EX 527 injection injected every time is preferably 0.2 mL.

The specific process for detecting the degree of inflammation of the prostate tissue of the EAP mouse is not particularly limited in the present invention, and can be performed according to the processes well known in the art.

According to the invention, Sirt1 inhibitor-EX 527 injection is injected into the abdominal cavity of an EAP mouse, the degree of inflammation of prostate tissues of the EAP mouse is detected, and whether the protection effect of melatonin on prostatitis can be inhibited by promoting the expression of NLRP3, ASC, caspase-1 and IL-1 beta is verified.

The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1

(1) Establishing an autoimmune prostatitis mouse model (EAP) by injecting a mixture of prostate antigen and complete Freund's adjuvant (volume ratio of 1:1) subcutaneously into non-obese diabetic mice (NOD) on days 0 and 28;

(2) preparing melatonin injection 8 mg/mL: dissolving 0.008g of melatonin in 0.05mL of dimethyl sulfoxide, and mixing the obtained solution (with the mass concentration of 0.16%) with 0.95mL of physiological saline to obtain a melatonin injection;

(3) injecting 0.2mL melatonin injection (once a day, the total injection amount is 80mg/kg/d each time) into the abdominal cavity of an EAP mouse from the 28 th day of subcutaneous immunization at 8-9 am every day until the mouse is sacrificed on the 42 th day; the degree of inflammation was assessed by histopathology, plantar pain measurement in EAP mice, peripheral blood ELISA, and then expression of NLRP3, ASC, caspase-1, IL-1 β, and Sirt1 in prostate tissue in EAP mice was examined by Western blotting and immunohistochemistry.

Example 2

(1) Establishing an autoimmune prostatitis mouse model (EAP) by injecting a mixture of prostate antigen and complete Freund's adjuvant (volume ratio of 1:1) subcutaneously into non-obese diabetic mice (NOD) on days 0 and 28;

(2) 8mg/mL of melatonin injection prepared in example 1 was used, while 0.5mg/mL of Sirt1 inhibitor-EX 527 injection was prepared: 0.0005g of EX527 was dissolved in 0.05mL of dimethyl sulfoxide, and the obtained solution (mass concentration: 0.1%) was mixed with 0.95mL of physiological saline to obtain an Sirt1 inhibitor-EX 527 injection;

(3) injecting melatonin injection into the abdominal cavity of an EAP mouse (once a day, the volume of each injection is 0.2mL, and the total injection amount is 80mg/kg/d each day) from the 28 th day of subcutaneous immunization at 8-9 am every day until the mouse is sacrificed on the 42 th day;

(4) from the day before the melatonin injection, the mice were injected with the Sirt1 inhibitor-EX 527 injection every other day into the abdominal cavity (once a day, the volume of each injection is 0.2mL, and the total injection amount is 5mg/kg per day), and the degree of inflammation of prostate tissue of EAP mice was examined.

Comparative example 1(Control group)

(1) Establishing an autoimmune prostatitis mouse model (EAP) by subcutaneously injecting a sterile normal saline and a complete Freund's adjuvant mixture (volume ratio of 1:1) into non-obese diabetic mice (NOD) on days 0 and 28;

(2) preparing a control group injection, namely mixing 0.05mL of dimethyl sulfoxide with 0.95mL of normal saline to obtain a dimethyl sulfoxide solution with the mass concentration of 5%;

(3) injecting 0.2mL of control group injection into the abdominal cavity of the EAP mice at 8-9 am every day from the 28 th day of subcutaneous immunization of the EAP mice until the mice are sacrificed at 42 th day; the degree of inflammation was assessed by histopathology, plantar pain measurement in EAP mice, peripheral blood ELISA, and then expression of NLRP3, ASC, caspase-1, IL-1 β, and Sirt1 in prostate tissue in EAP mice was examined by Western blotting and immunohistochemistry.

Comparative example 2(EAP + DMSO group)

(1) Establishing an autoimmune prostatitis mouse model (EAP) according to the method of the embodiment 1;

(2) preparing a control group injection, and mixing 0.05mL of dimethyl sulfoxide with 0.95mL of normal saline to obtain a dimethyl sulfoxide solution with the mass concentration of 5%;

(3) from the 28 th day of subcutaneous immunization, 8-9 am every day, 0.2mL of the control group injection of step (2) was intraperitoneally injected into EAP mice until the mice were sacrificed at day 42, the degree of inflammation was evaluated according to histopathology, plantar pain measurement in EAP mice, peripheral blood ELISA, and then expression of NLRP3, ASC, caspase-1, IL-1 β, and Sirt1 in prostate tissue in EAP mice was detected according to Western blotting and immunohistochemistry.

The result of the detection

In FIG. 1, A is a representative representation of histological staining of the prostate gland of the treated mice in example 1 and comparative examples 1-2 (wherein a + d represents a Control group (comparative example 1), b + e represents an EAP + DMSO group (comparative example 2), c + f represents an EAP + Mel group (example 1), a, b, c, original magnification:. times.100, bar:. mu.m; d, e, f, original magnification:. times.200, bar:. mu.m); as can be seen from a in fig. 1, melatonin reduced the degree of inflammatory changes in EAP prostate tissue, and the prostate tissue inflammatory changes in the EAP + Mel group mouse model were consistent with those in the Control group.

FIG. 1B is a graph showing a comparison of pathological change scores of prostate tissues of mice treated in example 1 and comparative examples 1 to 2; according to the pathological scoring standard, the pathological change scores of the three groups of prostate tissues are respectively: control group: 0.60 ± 0.24, EAP + DMSO group: 2.20 ± 0.20, EAP + Mel group: 1.00 +/-0.32.

In FIG. 1, C is a graph showing the difference between the test responses of the mice treated in example 1 and comparative examples 1-2 to pelvic pain: as can be seen from C in FIG. 1, the pot cavity pain response in the EAP + Mel group was significantly weaker than in the EAP + DMSO group when stimulated with 1.0g and 4.0g force, while the pain response was consistent in the control + DMSO and EAP + Mel group mice.

FIG. 1D is a graph showing effects of IFN-. gamma. (a), IL-17(b), and IL-1. beta. (c) on the expression level of inflammatory cytokines in the serum of mice treated in example 1 and comparative examples 1-2; from D in FIG. 1, the expression levels of IFN-. gamma., IL-17, and IL-1. beta. in the serum of mice in the EAP + Mel group were significantly reduced compared to those in the EAP + DMSO group (. times.P <0.001,. times.P <0.01,. times.P < 0.05).

As can be seen from fig. 1, melatonin relieves the changes in prostate inflammation in the EAP model, indicating that melatonin has a therapeutic effect on EAP.

FIG. 2 is an immunohistochemical analysis chart of the mouse prostate tissue treated in example 1 and comparative examples 1-2 (a, d and g are control groups; b, e and h are EAP + DMSO groups; c, f and i are EAP + Mel groups; original magnification:. times.200; bar is 50 μm), and it can be seen from FIG. 2 that the expression of NLRP3, ASC and caspase-1 in the prostate tissue of mice in EAP + Mel group is obviously reduced compared with the EAP + DMSO group, which shows that melatonin reduces the expression of NLRP3 inflammation-related protein in the prostate tissue of EAP mice, and that melatonin exerts the treatment effect on EAP by reducing the expression of NLRP3 inflammation-associated protein.

A in figure 3 is a result of western blot detection of the mice treated in example 1 and comparative examples 1-2, and A in figure 3 shows that the expression of NLRP3, ASC and caspase-1 in prostate tissues of mice in EAP + Mel group is obviously reduced compared with that in EAP + DMSO group, while the expression level of Sirt1 is obviously increased.

B, C, D, E and F in FIG. 3 are plots of the relative densities of NLRP3, ASC, caspase-1, IL-1 β and Sirt1 bands, respectively, versus the corresponding β -actin bands (. about.P <0.0001,. about.P <0.001,. about.P <0.01,. about.P < 0.05); from B, C, D, E and F in FIG. 3, it can be seen that in EAP, melatonin activates Sirt1 signaling pathway and decreases expression level of NLRP3 inflammation-related protein and IL-1 β, indicating that the therapeutic effect of melatonin on EAP may depend on activation of Sirt1 pathway to inhibit the expression of inflammatory body-related protein.

In FIG. 4, A is a representative representation of histological staining of the prostate gland of mice treated in examples 1-2 and comparative examples 1-2 (a, e are control + DMSO group (comparative example 1), b, f are EAP + DMSO group (comparative example 2), c, g are EAP + Mel group (example 1), d, h are EAP + Mel + EX527 group (example 2), a, b, c, d, original magnification:. times.100, bar:. mu.m; e, f, g, h, original magnification:. times.200, bar:. mu.m). As can be seen from A in FIG. 4, the inhibitor Sirt 1-EX 527 inhibits the therapeutic effect of melatonin on the EAP murine model, i.e., the changes in prostate tissue inflammation in mice of EAP + Mel + EX527 group are consistent with those in EAP + DMSO group.

FIG. 4B is a graph showing a comparison of pathological change scores of prostate tissues of mice treated in examples 1 to 2 and comparative examples 1 to 2; as shown in fig. 4B, the pathological changes of the four prostate tissues are scored as follows: 0.67. + -. 0.21 (control + DMSO), 2.17. + -. 0.31(EAP + DMSO), 0.83. + -. 0.31(EAP + Mel), 1.67. + -. 0.33(EAP + Mel + EX 527).

In FIG. 4, C is a graph showing the difference between the test responses of the mice treated in examples 1-2 and comparative examples 1-2 to pelvic pain; as can be seen from C in FIG. 4, the pelvic pain response was significantly stronger in the EAP + Mel + EX527 group than in the EAP + Mel group when stimulated with 1.0g and 4.0g force, while the pain response was consistent in the EAP + Mel + EX527 and EAP + DMSO group mice.

FIG. 4D is a graph showing the expression levels of IFN-. gamma. (a), IL-17(b), and IL-1. beta. (c) inflammatory cytokines in the serum of mice treated in examples 1-2 and comparative examples 1-2; from D in FIG. 4, the expression levels of IFN-. gamma., IL-17, and IL-1. beta. in the serum of mice in the EAP + Mel + EX527 group were significantly higher than those in the EAP + Mel group, and the expression levels were consistent with those in the EAP + DMSO group (. beta. P <0.0001,. beta. P <0.01,. beta. beta.);

as can be seen from the combination of FIG. 4, EX527 antagonizes the therapeutic effect of melatonin on EAP, and aggravates the severity of prostatitis, indicating that Sirt1 inhibitor-EX 527 can inhibit the protective effect of melatonin on EAP model, and aggravate the severity of prostatitis.

FIG. 5 is a graph of immunohistochemical analysis of prostate tissue of mice treated in examples 1-2 and comparative examples 1-2 (a, e and i are control + DMSO groups; b, f and j are EAP + DMSO groups; c, g and k are EAP + Mel groups; d, h and l are EAP + Mel + EX527 groups; original magnification:. times.200, bar 50 μm); as shown in FIG. 5, the expression of NLRP3, ASC and caspase-1 in prostate tissue of mice in EAP + Mel + EX527 group is obviously increased compared with that in EAP + Mel group, which indicates that EX527 increases the expression of NLRP3 inflammatory body associated protein in prostate tissue of melatonin-treated EAP, and indicates that EX527 antagonizes the therapeutic effect of melatonin on EAP probably acts by activating NLRP3 inflammatory bodies.

FIG. 6 shows the results of Western blot analysis of treated mice in examples 1-2 and comparative examples 1-2, and in FIG. 6, A shows that the expression levels of NLRP3, ASC and caspase-1 in prostate tissues of mice in EAP + Mel + EX527 group are obviously higher than those in EAP + Mel group, while the expression level of Sirt1 is obviously lower.

B, C, D, E and F in FIG. 6 are plots of the relative density of NLRP3, ASC, caspase-1, IL-1 β and Sirt1 bands versus the corresponding β -actin bands (. P <0.01,. P <0.05), respectively, and as can be seen from B, C, D, E and F in FIG. 6, EX527 increased the expression of NLRP3 inflammatory-related protein and IL-1 β and inhibited the expression of Sirt1 in melatonin-treated EAP, indicating that EX527 exerts an antagonistic effect on melatonin-treated EAP by activating NLRP3 corpuscle inflammation.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.