CN115645406A

CN115645406A - Application of liensinine in preparation of medicine for treating chronic prostatitis/chronic pelvic pain syndrome

Info

Publication number: CN115645406A
Application number: CN202211432163.8A
Authority: CN
Inventors: 李婷; 袁秋贞; 徐新雅; 王晓丽; 李博; 周春梅
Original assignee: Shaanxi Traditional Chinese Medicine Hospital
Current assignee: Shaanxi Traditional Chinese Medicine Hospital
Priority date: 2022-11-16
Filing date: 2022-11-16
Publication date: 2023-01-31

Abstract

The invention provides application of liensinine in preparation of a medicine for treating chronic prostatitis/chronic pelvic pain syndrome, and belongs to the field of medicines. The liensinine can improve abnormal urination symptoms of a CP/CPPS model mouse and relieve pain symptoms of the CP/CPPS model mouse, and the liensinine can treat chronic prostatitis/chronic pelvic pain syndrome.

Description

Application of liensinine in preparation of medicine for treating chronic prostatitis/chronic pelvic pain syndrome

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to application of liensinine in preparation of a medicine for treating chronic prostatitis/chronic pelvic pain syndrome.

Background

Prostatitis is one of the most common diseases of men, and accounts for about 1/3 of the total number of outpatients of urology andrology in China. Wherein, chronic Prostatitis/Chronic Pelvic Pain Syndrome (CP/CPPS) accounts for more than 90% of the total Prostatitis patients. Due to the characteristics of high morbidity, delayed and untreatable disease course, difficult cure, easy relapse and the like of CP/CPPS, the long-term illness has serious influence on the life quality of male patients, and simultaneously causes huge economic burden on public health services, and the CP/CPPS is one of public health problems which are concerned globally.

In view of the characteristics of multiple causes, multiple progression ways, inconsistent response to treatment and the like of CP/CPPS, the current treatment aiming at CP/CPPS has no unified standard. The main goals of CP/CPPS treatment are to relieve symptoms, including improving urination symptoms, relieving pain symptoms, and improving the quality of life of the patient. When a single medicament is used for treatment, a satisfactory treatment effect is difficult to obtain, so that a combined medicament mode is usually used for treatment clinically. Antibiotic, alpha ₁ Adrenergic receptor blocking agent, non-steroidal anti-inflammatory analgesic, are three most commonly used drugs for treating CP/CPPS at present.

However, when CP/CPPS is treated by combination medication, risk factors still exist and need to be comprehensively evaluated further due to rationality, effectiveness, safety and cost effectiveness. Therefore, the active ingredients which can improve the urination symptom of the patient and relieve the pain symptom of the patient are found, the action mechanism of the active ingredients is clarified, and the active ingredients have important guiding significance for developing novel CP/CPPS treatment medicines.

In the theory of traditional Chinese medicine, CP/CPPS belongs to the categories of stranguria, turbid semen, turbid white and the like, the clinical differentiation and the type mainly include damp-heat accumulation syndrome, qi stagnation and blood stasis syndrome, liver qi stagnation syndrome, kidney yin deficiency syndrome and the like, and the clinical manifestations are complex and are common in multiple syndromes. The traditional Chinese medicine for treating CP/CPPS is very rich, provides more choices for the treatment of CP/CPPS, and has more positive significance and advantages in the aspect of treating CP/CPPS.

The lotus plumule is derived from dried young leaves and radicles of mature seeds of lotus of Nymphaeaceae, is mainly produced in Hunan, hubei, fujian, jiangsu, zhejiang and other places, is a variety accepted by pharmacopoeia, and has the effects of clearing heart fire, tranquilizing mind, restoring normal coordination between heart and kidney, and arresting seminal emission and stopping bleeding. The liensinine is a dibenzyltetrahydroisoquinoline alkaloid separated from plumula Nelumbinis, and its molecular formula is C ₃₇ H ₄₂ N ₂ O ₆ The structure is shown as formula (1). Researches show that liensinine has various biological activities of resisting cancer, reducing blood pressure, resisting arrhythmia, resisting diabetes, protecting nerves, resisting inflammation, resisting oxidation and the like, but no research report about liensinine having CP/CPPS activity exists at present.

Disclosure of Invention

In view of the above, the invention aims to provide an application of liensinine in preparation of a medicine for treating chronic prostatitis/chronic pelvic pain syndrome, and provides a new application of liensinine.

In order to achieve the above purpose, the invention provides the following technical scheme:

the invention provides application of liensinine in preparation of a medicine for treating chronic prostatitis/chronic pelvic pain syndrome.

Preferably, the medicament comprises a clinically acceptable pharmaceutical formulation.

Preferably, the pharmaceutical preparation comprises tablets, capsules or granules.

The invention constructs a CP/CPPS model mouse by an antigen induction method. Bladder manometry experiments show that various urine kinetic parameters of the CP/CPPS model mouse are obviously changed compared with a blank control group, namely the CP/CPPS model mouse has obvious urination abnormity. After the liensinine is administrated by gastric lavage, the urodynamic parameter values of the CP/CPPS model mouse show improvement effects of different degrees, which are similar to the effects of the positive drug tamsulosin, and the liensinine is shown to be capable of improving the abnormal urination symptoms of the CP/CPPS model mouse.

On the other hand, hot plate analgesia experiments show that compared with a blank control group mouse, the CP/CPPS model mouse has shortened heat-shrinkable foot latency; the Von-Frey acupuncture pain threshold test shows that the CP/CPPS model mouse has an increased acupuncture positive reaction frequency compared with a blank group mouse, and the CP/CPPS model mouse shows obvious pain threshold reduction and pain sensation sensitivity. After liensinine is administrated by gastric lavage, the hot plate retention time of a CP/CPPS model mouse is prolonged, the pain threshold value is increased, and the positive reaction frequency of the Von-Frey acupuncture is reduced, which shows that the liensinine can relieve the pain symptom of the CP/CPPS model mouse.

Compared with the prior art, the invention has the beneficial effects that:

the liensinine has low toxicity to human body and strong applicability. Therefore, the liensinine can be used for preparing products for treating CP/CPPS, the invention not only provides a new application of the liensinine, but also provides a new candidate compound for discovering CP/CPPS curative substances capable of improving abnormal urination and relieving pain symptoms of the abnormal urination.

Drawings

FIG. 1 is a graph showing HE staining of prostate tissue of mice in each group, wherein A is a blank control group, B is a sham operation group, and C is a model control group;

FIG. 2 is a flow chart of a mouse urodynamics determination experiment;

fig. 3 is a graph of bladder pressure (n = 8) of each group of mice after administration of liensinine, wherein a is a blank control group, B is a sham group, C is a CP/CPPS model group, D is a tamsulosin-treated group (0.03 mg/kg), E is a liensine low dose treated group (15 mg/kg), F is a liensine medium dose treated group (30 mg/kg), G is a liensine high dose treated group (60 mg/kg), H is a time histogram of urination intervals of each group of mice, I is a histogram of maximum urination pressure of bladder of each group of mice, and J is a histogram of threshold urination threshold pressure of bladder of each group of mice, with the result expressed as mean ± s.e.m, <0.001 compared to the blank control group; compared to the CP/CPPS model group, # P <0.05, # P <0.01, # P <0.001.

FIG. 4 is a graph showing the improvement effect of liensinine on the pain symptoms of a CP/CPPS model mouse (n = 8), wherein A is a graph showing the results of a hot plate analgesic test of the mouse, B is a graph showing the results of a mechanical pain domain test of the mouse by a Von-Frey acupuncture method, and the results are expressed as mean + -s.e.m,. P is less than 0.01 and P is less than 0.001 compared with a blank control group; compared to the CP/CPPS model group, # P <0.05, # P <0.01.

Detailed Description

The invention provides application of liensinine in preparation of a medicine for treating chronic prostatitis/chronic pelvic pain syndrome. In the present invention, the medicament preferably comprises a clinically acceptable pharmaceutical preparation. In the invention, the pharmaceutical preparation preferably comprises tablets, capsules or granules, the preparation method of the dosage form is not particularly limited by the invention, and the preparation can be carried out by a person skilled in the art according to the conventional dosage form, the content of the liensinine in the dosage form is not particularly limited by the invention, and the person skilled in the art can carry out preparation according to the content of active substances in the conventional medicine.

The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.

Example 1

1. Test method

1. Effect of liensinine on improving abnormal urination of CP/CPPS model mice

1.1 antigen-induced method for constructing CP/CPPS model mouse

1.1.1 preparation of prostate antigen

20 male Wister rats of SPF grade 180-200 g were purchased from Experimental animals technology GmbH, wei Tony, beijing. After anesthesia with 4% chloral hydrate, the lower abdomen was incised to obtain the prostate tissue of the rat. After removing the excess adipose tissue, the prostate was weighed and washed with physiological saline. An equal weight of 0.01mmol/LpH solution with a value of 7.2 (containing 0.5% Triton X-100 and 0.1mM protease inhibitor) was added. The tissue was homogenized on ice, centrifuged at 10000rpm at 4 ℃ for 30min, and the supernatant was aspirated. After the protein concentration of the supernatant was measured by the BCA protein concentration detection kit, it was quantified to 50mg/mL with PBS solution and stored at-20 ℃ for further use.

1.1.2 construction of mouse CP/CPPS model by immunization

Male C57BL/6J mice, 7-8 w, were purchased from Experimental animals technology, inc. of Viton, beijing. Feeding the seeds in an SPF animal house at the indoor temperature of 20-25 ℃; the humidity is 50% -60%; bedding was changed 3 times per week with food and water added.

Two syringes are connected to two ends of the tee respectively, wherein one syringe extracts the supernatant of the Wister rat prostate, and the other syringe extracts the same volume of Freund's Complete adjuvant (CFA), and the mixture is emulsified on ice. When the mixed solution is milky white and dropped into water, the mixed solution is in a water-in-oil state, which indicates complete emulsification. Mice were immunized with the mixture of rat prostate extract supernatant plus CFA when emulsified completely, and the primary immunization was recorded as 0d. Each mouse was injected subcutaneously at multiple sites, including bilateral footpads (0.025 mL/side), the tail root (0.050 mL) and the shoulder (0.050 mL). At 28d, the mixed solution is used again to immunize the mice subcutaneously, the dosage and the method are consistent with those of the primary immunization, the mice in the sham operation group are injected with physiological saline with the same volume subcutaneously, and the mice are subjected to gastric lavage administration at 32 d.

1.2 groups of Experimental animals

40 mice in the CP/CPPS model group are taken and randomly divided into five groups, wherein the five groups are respectively as follows: tamsulosin treatment group (0.03 mg/kg), liensinine low dose treatment group (15 mg/kg), liensinine medium dose treatment group (30 mg/kg), liensinine high dose treatment group (60 mg/kg), CP/CPPS model control group (normal saline). The medicines in each group are administered by intragastric administration for 10 days.

TABLE 1 grouping and administration of laboratory animals

Grouping of laboratory animals	Dosage of drug
		Blank Control group (Control)	-
False operation group (Sham)	-
		Model control group (Model)	Physiological saline
Tamsulosin treatment group (Tam)	0.03mg/kg
		Lotus seed plumule alkali low dose treatment group (Lien-L)	15mg/kg
Lotus seed plumule alkali low dose treatment group (Lien-M)	30mg/kg
		Lotus seed plumule alkali low dose treatment group (Lien-H)	60mg/kg

1.3 measurement of urodynamic parameters of mice

Prior to cystometry, mice were fasted for 24h, maintaining free access to water. The mice were removed and anesthetized with 10% chloral hydrate by abdominal injection (0.1 mL/10 g), placed on a hot plate for animal experiments, set at 37 deg.C, and subjected to abdominal incision in the supine position to expose bladder tissue. The vein indwelling needle was punctured into the bladder and carefully fixed. The other end of the indwelling needle is respectively connected with a micro-injection pump and a pressure transducer through a three-way pipe, physiological saline preheated at 37 ℃ is continuously filled into the urinary bladder of the mouse at 2.0mL/h in the injection pump, and the pressure transducer converts a pressure signal into an electric signal through a signal amplification system (BL 420S) and transmits the electric signal to a computer screen. Normal saline is infused for 0.5-1 h at room temperature, after the mouse bladder micturition reaction is stable, bladder pressure value change is recorded, 1h is observed, 3-5 complete micturition periods in an observation period are taken, pressure indexes are recorded, and parameters are calculated: voiding Interval Time (VIT), the average interval time between urination activities; maximum Micturition Pressure (MP) of the bladder, i.e. the peak pressure in the bladder at the time of urination; bladder urination threshold pressure (MTP), the threshold pressure at which a micturition response is just initiated and elicited.

1.4 prostate index calculation

After the mice were sacrificed by cervical dislocation, the prostate tissue of the mice was dissected and isolated. Excess fat and connective tissue on the tissue surface were carefully removed, excess fluid on the prostate surface was blotted with filter paper and immediately weighed wet. Prostate index (stat index, PI) = wet weight of prostate (mg)/body weight of mouse (g) was calculated.

1.5 histopathological examination of prostate

The morphology of the prostate tissue of each group of mice was observed by HE staining. After the mouse prostate tissue was weighed, it was fixed with 4% paraformaldehyde. After fixation, dehydrating with ethanol solution with gradient concentration, adding into xylene for transparency, taking out tissue, soaking in wax, embedding, cooling on ice, and solidifying wax block. After slicing by a slicer, baking the slices in an oven for standby. The sections were removed, dewaxed in xylene solution and subsequently immersed in a concentration gradient of ethanol solution. Taking out tap water, washing for 10s, and dyeing in hematoxylin dyeing solution for 5min. Excess dye was rinsed off with tap water and the section was observed to turn blue and stopped. And then placing the slices into eosin dye solution for dyeing for 2min, taking out the slices, and sequentially placing the slices into ethanol solution with gradient concentration for dehydration. And (3) clearing the xylene solution for three times, wiping off the redundant xylene solution around the sliced tissues, dripping neutral resin, and sealing the slices by a cover glass. The prostate tissue morphology is observed under the light microscope, and the photographing record is carried out.

2. Relieving effect of liensinine on pain symptoms of CP/CPPS model mice

2.1 Hot plate analgesia assay in mice

Adjusting a hot plate analgesia instrument to control the temperature to be 55 +/-0.5 ℃, preheating the hot plate for 10min, then placing the mouse on the hot plate, wherein the time required by the mouse from the placement to the occurrence of licking is the heat-shrinkable foot latency period, namely the pain threshold value, of the mouse. 40 screened qualified (licking time is less than 5s or more than 30s or skipper abandoning) mice are randomly divided into 5 groups, each group comprises 8 mice, and the mice are respectively a blank control group, a neferine high-dose group, a neferine medium-dose group and a neferine low-dose group (60 mg/kg, 30mg/kg and 15 mg/kg) and a CP/CPPS model control group. The pain threshold value of the model CP/CPPS mouse is determined at the 32 nd construction stage, and the average value of the two times is taken as the initial pain threshold value. Mice in the administration group are subjected to intragastric administration for 10 days continuously, and the model control group is subjected to physiological saline with the same volume. At 0d, 5d and 10d post-dose (i.e., at 32d, 37d and 42d post-molding), the pain threshold was determined for each group of mice, the data was recorded and the mean value for each group was calculated.

2.2 mechanical pain threshold test in mice

The change of the mechanical pain threshold value of the mice after the liensinine administration is determined by a Von-Frey acupuncture method. The mice were placed on a stainless steel console and acclimated for half an hour to be quiet, and the skin in the center of the scrotum was stimulated with a series of Von-Frey filaments from below the console. The bending degree of the fiber filaments is kept the same as much as possible in the stimulation operation process, and the stimulation parts are the same each time, so that the experimental error is reduced. Mice were stimulated sequentially from small to large with different weight of filaments, each filament staying 1-2s apart 5s, for a total of 5 determinations, depending on the load bearing range of the Von-Frey filaments. When the mouse has subjective pain and actively moves the scrotum, licks or scratches the scrotum immediately and the abdomen contracts or jumps sharply, the mouse is considered to have a positive reaction of mechanical stimulation. The average percentage of positive mechanical stimulation responses was determined for each group of mice at 32 days of CP/CPPS model mouse construction, at 10 days of gavage (15 mg/kg, 30mg/kg and 60mg/kg liensinine) (e.g., 40% positive responses were observed after 5 stimulations, 2 times).

2. Results of the experiment

Successful construction of CP/CPPS model mouse

As shown in table 2, there was no significant difference in the weight and prostate wet weight average of sham mice compared to the control of the blank group (P > 0.05). While the prostate weight of the model group mice increased and the prostate index significantly increased (P < 0.05), suggesting that the CFA mixed injection induced prostate tissue lesions in the mice. In addition, whether the model is successfully made or not is preliminarily judged by an HE staining method, and as shown in figure 1, a CP/CPPS model mouse is successfully constructed by an antigen induction method. The interstitial tissues of the prostate gland of the mice in the model group have obvious inflammatory cell infiltration, the acini have irregular arrangement and edema, necrosis and shedding with different degrees, and the prostate tissues of the mice in the sham operation group and the mice in the blank control group are orderly arranged, and the cell boundary is clear.

Table 2 prostate index comparison of groups of mice (n = 8)

Note: p <0.001 compared to sham group.

2. Effect of liensinine on improving abnormal urination of CP/CPPS model mice

After the constructed CP/CPPS model mice were administered in groups, the bladder pressure curves of the mice in each group were measured and the values of the urodynamic parameters were calculated (FIG. 2). The measurement results are shown in fig. 3, and the values of urodynamic parameters of the mice in the sham operation group are not significantly different (P > 0.05) compared with the mice in the blank control group. The values of various urodynamic parameters of the CP/CPPS model mouse are obviously changed (figure 3), such as prolonged micturition interval, increased maximum micturition pressure of the bladder and increased threshold micturition pressure. After tamsulosin treatment, the abnormal urination symptoms of the CP/CPPS model mouse are obviously improved, such as shortening of the urination interval of the mouse, increase of the maximum urination pressure of the bladder and increase of the urination threshold pressure. Similarly, the urodynamic parameter values of the CP/CPPS model mouse show different degrees of improvement after 60mg/kg of liensinine administration, which shows that the liensinine can improve the abnormal urination symptoms of the CP/CPPS model mouse.

3. Effect of liensinine on improving pain symptoms of CP/CPPS model mice

The heat-shrinkable foot latency of the mice was recorded by using a hot plate stimulator, and as a result, as shown in fig. 4A, the residence time of the hot plate was shortened and the pain threshold was reduced to 7.8 ± 3.7s in the CP/CPPS model group at the 32d (0 d after administration) of the model group, as compared with the blank control group. With the extension of the molding time, the pain threshold of the model group mice further decreases, and the residence time of the hot plate at 42d is 2.9 +/-3.2 s. After liensinine treatment, compared with the model group, the hot plate residence time of mice in the administration group is prolonged to different degrees, in particular, the hot plate residence time of the mice is obviously prolonged at 5d and 10d (37 d and 42d of modeling) when 60mg/kg of neferine is administered, the hot plate residence time is respectively 7.5 +/-3.2 s and 8.0 +/-3.9 s, the pain threshold of the mice is increased, and the difference has statistical significance. When the mechanical pain threshold of the mice is further detected by the Von-Frey acupuncture method, the result is shown in fig. 4B, and the positive reaction frequency detected by the model group mice is remarkably increased to 59.3% + -11.2% (P < 0.05) at the 42d of modeling. After the liensinine of 60mg/kg is used for gastric perfusion, the positive reaction frequency of the mice in the administration group is obviously reduced and is only 35.6% +/-13.7% (P < 0.05). Hot plate analgesia experiments and Von-Frey acupuncture experiments show that liensinine has an improvement effect on the pain symptoms of CP/CPPS model mice.

In conclusion, the invention discloses the application of liensinine in the preparation of CP/CPPS treatment medicines. The invention discovers the improvement effect of liensinine on CP/CPPS urination abnormity and the alleviation effect on CP/CPPS pain symptoms for the first time. The invention provides new selection and thinking for the treatment of CP/CPPS at present and also makes a contribution to the development of the technical field. The invention is a compound with a definite chemical structure, is convenient for preparing modern dosage forms, and has the potential of becoming a drug for treating CP/CPPS.

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.

Claims

1. The use of liensinine in preparing medicine for treating chronic prostatitis/chronic pelvic pain syndrome is provided.

2. The use of claim 1, wherein the medicament comprises a clinically acceptable pharmaceutical formulation.

3. Use according to claim 2, wherein the pharmaceutical formulation comprises a tablet, capsule or granule.