Application of compound lysimachia christinae hance preparation in preparation of medicine for preventing and treating chronic pelvic pain syndrome

Abstract

The invention belongs to the technical field of biological medicine, and discloses application of a compound lysimachia christinae hance preparation in preparation of a medicine for preventing and treating chronic pelvic pain syndrome.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and discloses the application of compound Lysimachia christinae preparation in the preparation of drugs for the prevention and treatment of chronic pelvic pain syndrome. Background Technology

[0002] Chronic pelvic pain syndrome (CPPS) refers to a group of diseases characterized by non-periodic, persistent pelvic pain (including abdominal wall, pelvic organs, lumbosacral region, and perineum) lasting ≥6 months, with pain severity leading to functional impairment or requiring drug / surgical treatment. Its core pathological mechanism involves the interaction of inflammation, nerve sensitization, blood flow obstruction, and psychological factors, with etiologies encompassing multiple systems including gynecology, urology, digestion, and musculoskeletal diseases. The core symptom is persistent dull or aching pain, primarily in the lower abdomen and lumbosacral region, which worsens with prolonged standing, exertion, or menstruation, and is relieved by lying down. Western medicine primarily uses ibuprofen and celecoxib to relieve mild to moderate pain, but long-term use requires caution regarding peptic ulcers. Traditional Chinese medicine mainly uses herbal remedies such as Sanjie Zhentong capsules, which have a slower onset of action. CPPS is a very common urological disease in clinical practice, but its etiology and pathogenesis remain unclear, leading to difficulties in its treatment. Studies have shown that CPPS patients have increased secretion of inflammatory cytokines and upregulated expression of chemokines, which in turn induce related immune responses. This suggests that CPPS may be an autoimmune disease centered on the production of inflammatory factors. Compound Lysimachia christinae granules are a widely used traditional Chinese medicine in clinical practice, showing significant efficacy in treating urinary tract stones and urinary tract infections. However, there are currently no studies exploring the therapeutic effects of Compound Lysimachia christinae granules on CPPS. Summary of the Invention

[0003] This invention relates to the application of compound Lysimachia christinae preparations in the preparation of drugs for the prevention and treatment of chronic pelvic pain syndrome.

[0004] In some embodiments, the active ingredient of the preparation is prepared from 210-220 parts of Lysimachia christinae, 105-110 parts of Plantago asiatica, 105-110 parts of Pyrrosia lingua, and 50-55 parts of corn silk.

[0005] In some embodiments, the active ingredient of the preparation is prepared from 218 parts of Lysimachia christinae, 109 parts of Plantago asiatica, 109 parts of Pyrrosia lingua, and 54.5 parts of corn silk.

[0006] In some embodiments, the formulation preparation method is as follows:

[0007] Take Desmodium styracifolium, Plantago asiatica, Pyrrosia calvata, and Cornus officinalis. For these four herbs, Desmodium styracifolium, Plantago asiatica, and Cornus officinalis are decocted in water two to three times. For the first time, add 7 - 8 times the amount of water and decoct for 2 - 3 hours; for the second time, add 5 - 6 times the amount of water and decoct for 1 - 2 hours. Combine the decoctions, filter, and concentrate the filtrate to a clear paste with a relative density of 1.16 - 1.22 (70°C). Pyrrosia calvata is decocted in water two to three times. For the first time, add 7 - 8 times the amount of water and decoct for 2 - 3 hours; for the second time, add 5 - 6 times the amount of water and decoct for 1 - 2 hours. Combine the decoctions, filter, and concentrate the filtrate to a clear paste with a relative density of 1.16 - 1.22 (70°C). Let it cool, add 1.5 - 2 times the volume of ethanol, stir well, let stand for 24 - 30 hours, take the supernatant, recover the ethanol, concentrate to an appropriate amount, mix with the above clear paste, and continue to concentrate to a clear paste with a relative density of 1.16 - 1.22 (70°C). Dry and pulverize to obtain the product.

[0008] In some embodiments, the preparation method of the preparation is as follows:

[0009] Take Desmodium styracifolium, Plantago asiatica, Pyrrosia calvata, and Cornus officinalis. For these four herbs, Desmodium styracifolium, Plantago asiatica, and Cornus officinalis are decocted in water twice. For the first time, add 8 times the amount of water and decoct for 2 hours; for the second time, add 6 times the amount of water and decoct for 1 hour. Combine the decoctions, filter, and concentrate the filtrate to a clear paste with a relative density of 1.16 - 1.22 (70°C). Pyrrosia calvata is decocted in water twice. For the first time, add 8 times the amount of water and decoct for 2 hours; for the second time, add 6 times the amount of water and decoct for 1 hour. Combine the decoctions, filter, and concentrate the filtrate to a clear paste with a relative density of 1.16 - 1.22 (70°C). Let it cool, add 1.5 times the volume of ethanol, stir well, let stand for 24 hours, take the supernatant, recover the ethanol, concentrate to an appropriate amount, mix with the above clear paste, and continue to concentrate to a clear paste with a relative density of 1.16 - 1.22 (70°C). Dry and pulverize to obtain the product. Detailed implementation manners

[0010] The present invention provides an application of a compound Desmodium styracifolium preparation in the preparation of a drug for preventing and treating chronic pelvic pain syndrome, wherein the preparation, its active ingredients are prepared from 218 parts of Desmodium styracifolium, 109 parts of Plantago asiatica, 109 parts of Pyrrosia calvata, and 54.5 parts of Cornus officinalis.

[0011] I. Experimental materials

[0012] 1. Experimental animals

[0013] Male Wistar rats, SPF grade, 8 weeks old, weighing 180 - 200 g, provided by the Experimental Animal Center of Guangxi Medical University, experimental animal production license number: SCXK Gui 2020 - 0003. The animals are raised in an SPF - grade animal laboratory with sufficient food and water provided.

[0014] 2. Experimental drugs and reagents

[0015] The drug used in this invention is a dry extract powder, batch number: 2501001. This sample was provided by Nanjing Lianfang Traditional Chinese Medicine Technology Co., Ltd., and each 1g of dry extract powder is equivalent to 19.6g of medicinal slices.

[0016] II. Experimental Methods

[0017] 1. Preparation of Compound Lysimachia christinae Preparation

[0018] Take 218 parts of Lysimachia christinae, 109 parts of Plantago asiatica, 109 parts of Pyrrosia lingua, and 54.5 parts of corn silk. For the four herbs, decoct Lysimachia christinae, Plantago asiatica, and corn silk twice with water. For the first decoction, add 8 times the amount of water and decoct for 2 hours; for the second decoction, add 6 times the amount of water and decoct for 1 hour. Combine the decoctions, filter, and concentrate the filtrate to a clear extract with a relative density of 1.16–1.22 (70℃). For the Pyrrosia lingua, decoct twice with water. For the first decoction, add 8 times the amount of water and decoct for 2 hours; for the second decoction, add 6 times the amount of water and decoct for 1 hour. Combine the decoctions, filter, and concentrate the filtrate to a clear extract with a relative density of 1.16–1.22 (70℃). Cool, add 1.5 times the volume of ethanol, stir well, let stand for 24 hours, take the supernatant, recover the ethanol, concentrate to an appropriate volume, mix with the above clear extract, and continue to concentrate to a clear extract with a relative density of 1.16–1.22 (70℃). Dry, pulverize, and obtain the final product.

[0019] 2. Establishment of CPPS Model

[0020] A CPPS rat model was established using male Wistar rats. The specific procedures were as follows: Rats were anesthetized via intraperitoneal injection of 4% pentobarbital (40 mg / kg). After adequate anesthesia, the surgical site was disinfected and draped. After removing hair from the skin surface, the skin and muscle layer were separated from the left lower abdomen to expose the bladder. The two lateral lobes of the prostate were then separated and exposed from both sides of the bladder. The lateral lobes of the prostate were gently clamped and fixed with toothless forceps. 50 μL of CFA (Complete Freund's Adjuvant) was injected into each lateral lobe to induce aseptic inflammation of the prostate. Strict aseptic technique was maintained throughout the entire process. After injection, the muscle layer and skin were sutured, and the abdomen was closed. Postoperatively, 80,000 units of penicillin were injected intraperitoneally to prevent infection.

[0021] 3. Grouping and administration of experimental animals

[0022] Sixty rats were randomly divided into six groups: blank control group, model control group, low-dose group, medium-dose group, high-dose group, and positive control group.

[0023] Blank control group: Rats in this group did not undergo surgery. Starting from the second day after surgery, they were given an equal amount of sterile saline by gavage daily.

[0024] Model control group: The rats in this group were modeled with CPPS using the above method. Starting from the second day after the operation, they were given an equal amount of sterile saline by gavage every day.

[0025] Low-dose group: The rats in this group were modeled with CPPS using the above method. Starting from the second day after the operation, they were given compound Lysimachia christinae dry extract powder suspension by gavage (0.08g / kg / d) every day.

[0026] Medium-dose group: Rats in this group underwent CPPS modeling using the above method. Starting from the second day after surgery, they were given compound Lysimachia christinae dry extract powder suspension by gavage daily (0.16g / kg / d).

[0027] High-dose group: Rats in this group underwent CPPS modeling using the above method. Starting from the second day after surgery, they were given compound Lysimachia christinae dry extract powder suspension by gavage daily (0.32g / kg / d).

[0028] Positive drug group: Rats in this group underwent CPPS modeling using the above method. Starting from the second day after surgery, they were given pregabalin suspension by gavage daily (30 mg / kg / d).

[0029] Compound Lysimachia christinae extract powder and pregabalin were prepared into a suspension using physiological saline. The suspension was placed in a constant-temperature water bath beforehand to ensure complete dissolution of the drug and saline solution. The suspension was shaken well before administration to ensure accurate dosage. The rats were weighed weekly to adjust the dosage, and the administration was performed via gavage for a total of 30 days.

[0030] 4. Tissue sampling

[0031] After the final gavage, the rats were weighed and their weight recorded. Following mechanical pain threshold testing, the rats were euthanized by decapitation, and the lumbar enlargement segment of the spinal cord was rapidly removed on ice and stored at -80°C. Simultaneously, the rat prostate tissue was quickly and completely removed, rinsed in pre-cooled physiological saline, and carefully dissected and removed fat and other tissues. The wet weight of the prostate tissue was measured. A portion of the tissue was used for ELISA and immunoprotein detection, and the remaining prostate tissue was fixed in 4% paraformaldehyde and prepared into paraffin sections.

[0032] 5. Detection indicators

[0033] 5.1. Pain Behavioral Testing

[0034] Paw withdrawal threshold (PWT) determination: Rats were placed in a translucent plexiglass cage, and the mid-sole of the hind limb paw was stimulated with a series of standardized fine fibers (Von Frey fibers). Slight bending of the cilia was used as the standard for full force application, causing the fibers to bend into an S-shape (representing a mechanical stimulus of 1-50g), lasting 6-8 seconds. The paw withdrawal response was observed. Von Frey fibers were used sequentially in weight order (2, 4, 6, 8, 10, 15, 26g), with each stimulus lasting 2 seconds, followed by a 15-second interval, for 5 consecutive repetitions. The lowest weight of Von Frey fiber required to elicit 3 / 5 leg raises was defined as the PWT.

[0035] 5.2. Tissue Damage and Inflammation Detection

[0036] Prostate index detection: The prostate index is a significant indicator of prostate inflammation, and prostate weight increases as inflammation progresses. Record the rat's own body weight and total wet weight of prostate tissue. Prostate index = total wet weight of prostate tissue / rat body weight (unit: g / 100g).

[0037] Histological evaluation of the prostate: Prostate tissue was fixed in 4% neutral paraformaldehyde and embedded in paraffin. 5μm sections were stained with hematoxylin and eosin (HE); each slide was measured 5 times, with 5 slides per group. The degree of inflammation in the rat prostate was graded using a four-point scale, from 0 to 3. 0: No obvious inflammation; 1: Single inflammatory cell present; 2: Confluent sheet-like inflammatory cells present but without tissue destruction or lymphoid nodules / follicle formation; 3: Obvious sheet-like inflammatory cells accompanied by tissue destruction or lymphoid nodules / follicle formation of inflammatory cell clusters.

[0038] Detection of inflammatory factors in prostate tissue: An enzyme-linked immunosorbent assay (ELISA) with double antibodies was used to measure inflammatory factors in prostate tissue. Antibodies against tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β) were pre-coated on an ELISA plate. After adding standards and the sample to be tested, TNF-α, IL-1β, and IL-6 in the sample bound to the antibodies on the solid-phase carrier. After incubation, unbound samples were washed away and bound to the added detection antibodies, forming a double-sandwich antibody. After washing away excess detection antibodies, a chromogenic substrate was added. The levels of TNF-α, IL-1β, and IL-6 in the sample were positively correlated with the color intensity of the chromogenic substrate reaction. Specific experimental procedures must be strictly followed according to the ELISA kit instructions.

[0039] 5.3. Detection of Oxidative Stress Indicators

[0040] The tissue mass (g) to extraction liquid volume (ml) was homogenized thoroughly under ice bath conditions at a ratio of 1:5 to 1:10. The homogenate was centrifuged at 8000g and 4℃ for 10 min, and the supernatant was collected and placed in an ice bath for analysis. The expression of malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px) was detected strictly according to the kit instructions.

[0041] 5.4. Detection of expression of neurosensitization marker proteins (TRPV1, p-NF-κB(p-p65))

[0042] Total protein was extracted from the dorsal horn tissue of the lumbar enlargement segment of the spinal cord. After SDS-PAGE electrophoresis, transfection, and incubation with primary and secondary antibodies, chemiluminescence, development, and fixation were performed according to the ECL kit instructions. The grayscale of each band was measured using the Image-J software image analysis system. The target bands TRPV1 and p-NF-κB (p-p65) were compared with the internal control β-actin to correct for experimental errors in protein quantification and sample loading.

[0043] III. Experimental Results

[0044] 1. Behavioral testing for pain

[0045] As shown in Table 1, compared with the blank control group, the pain threshold of the model control group rats was significantly reduced (P<0.05), indicating that the CPPS rat model was successfully established, while the high-dose group and the positive drug group could significantly increase the pain threshold (P<0.05).

[0046] 2. Tissue damage and inflammation detection

[0047] (1) Prostate weight and prostate index

[0048] Prostate weight and index are good indicators of the degree of prostate congestion and edema, especially the prostate index, which more accurately reflects the degree of prostate inflammation after being calibrated for body weight. As shown in Table 1, compared with the blank control group, the prostate index of the model control group rats was significantly increased (P<0.05), indicating that the CPPS rat model was successfully established, and the medium and high dose groups and the positive control group could significantly reduce prostate inflammation (P<0.05).

[0049] Table 1: Effects of the present invention on prostate index and pain threshold

[0050]

[0051] (2) Histological evaluation of the prostate

[0052] In the blank control group, the glandular epithelial cells were neatly arranged without hyperplasia or atrophy, the glands were tightly packed, and no obvious pathological changes such as edema, exudation, or inflammation were observed in the stroma. In the model control group, the glandular epithelial cells were neatly arranged without hyperplasia or atrophy, but some glandular epithelial cells showed inflammatory cell infiltration, the glands were loosely arranged, and pathological changes such as inflammatory cell infiltration and stromal hyperplasia were observed in the stroma. In the low-dose group, the glandular epithelial cells were neatly arranged without hyperplasia or atrophy, the glands were loosely arranged, and edema with inflammatory cell infiltration was observed in the stroma. In the medium-dose and high-dose groups, the glandular epithelial cells were neatly arranged without hyperplasia or atrophy, the glands were tightly packed, and no obvious pathological changes such as edema, exudation, or inflammation were observed in the stroma. The specific scores are shown in Table 2. Compared with the blank control group rats, the inflammation score of the model control group was significantly increased (P<0.01), indicating that the CPPS rat model was successfully established. The compound Lysimachia christinae extract powder and the positive control group can significantly reduce prostate inflammation (low dose group: P<0.05; medium and high dose and positive control group: P<0.01).

[0053] Table 2: Effects of the present invention on prostate tissue inflammation score

[0054]

[0055] (3) Detection of inflammatory factor levels in prostate tissue

[0056] As shown in Table 3, compared with the blank control group, the levels of inflammatory factors IL-1β, IL-6, and TNF-α in prostate tissue were significantly increased in the model control group (IL-1β and IL-6: P < 0.01; TNF-α: P < 0.05). After treatment, the levels of specific inflammatory factors in each treatment group decreased compared with the model control group. Among them, the level of IL-1β decreased in all treatment groups with statistical differences (low and medium dose groups: P < 0.05; high dose group and positive control group: P < 0.01); the level of IL-6 was significantly decreased in the medium and high dose groups and the positive control group (medium and high dose groups: P < 0.05; positive control group: P < 0.01); the level of TNF-α was significantly decreased only in the high dose group and the positive control group (P < 0.05).

[0057] Table 3: Effects of the present invention on the levels of inflammatory factors in prostate tissue

[0058]

[0059]

[0060] 3. Detection of oxidative stress indicators

[0061] Increased oxidative stress levels are often accompanied by MDA accumulation, decreased SOD content, and reduced GSH-Px activity; therefore, these three factors are widely used to assess tissue oxidative stress levels. In inflammatory environments, disruption of oxidative stress homeostasis often leads to the accumulation of oxygen free radicals, resulting in oxidative stress. As shown in Table 4, the MDA concentration in the prostate tissue of the model control group was higher than that in the blank control group, while SOD and GSH-Px activities were lower, with statistically significant differences (MDA and GSH-Px: P < 0.05; SOD: P < 0.01). This phenomenon was significantly reversed in the medium- and high-dose groups and the positive control group.

[0062] Table 4: Effects of this invention on oxidative stress levels

[0063]

[0064] 4. Detection of expression of neurosensitization marker proteins

[0065] Activation of the voltage-gated calcium channel activator TRPV1 enhances the pain response, and its expression is upregulated in various inflammatory and neuropathic pain models. As shown in Table 5, compared with the blank control group, the protein expression level of TRPV1 in the model control group was significantly increased (P < 0.01); however, compared with the model control group, its expression level was significantly inhibited after the intervention of compound Lysimachia christinae extract powder and positive control drug (low and medium dose groups: P < 0.05; high dose group and positive control drug group: P < 0.01).

[0066] Nuclear factor-κB (NF-κB) is a central mediator of inflammation, regulating the expression of many inflammatory signaling genes. Within the NF-κB subunit, p65 is a key member of the classical NF-κB pathway. The number of nuclear translocations in the activated state of p-p65 reflects the stage of inflammation, while reduced p-p65 expression indicates inhibition of NF-κB signaling pathway activation. As shown in Table 5, compared to the blank control group, the protein expression level of p-NF-κB (p-p65) in the model control group was significantly increased (P < 0.01); however, compared to the model control group, the expression level of p-NF-κB (p-p65) was significantly decreased in the medium-dose group, high-dose group, and positive control group (P < 0.01).

[0067] Table 5: Effects of this invention on the expression levels of neurosensitization marker proteins

[0068] Note: In the table above, compared with the blank control group, # P<0.05, ## P<0.01; compared with the model group, * P<0.05, **P<0.01。

Claims

1. Application of compound Lysimachia christinae preparation in the preparation of drugs for the prevention and treatment of chronic pelvic pain syndrome.

2. The compound Lysimachia christinae preparation as described in claim 1, wherein the active ingredients of the preparation are prepared from 210-220 parts of Lysimachia christinae, 105-110 parts of Plantago asiatica, 105-110 parts of Pyrrosia lingua, and 50-55 parts of corn silk.

3. The compound Lysimachia christinae preparation as described in claim 2, wherein the active ingredients of the preparation are prepared from 218 parts of Lysimachia christinae, 109 parts of Plantago asiatica, 109 parts of Pyrrosia lingua, and 54.5 parts of corn silk.

4. The compound Lysimachia christinae preparation according to any one of claims 1-3, wherein the preparation method is as follows: Take the following four herbs: Lysimachia christinae, Plantago asiatica, Pyrrosia lingua, and Zea mays silk. Decoct Lysimachia christinae, Plantago asiatica, and Zea mays silk two to three times with water. For the first decoction, add 7 to 8 times the amount of water and decoct for 2 to 3 hours. For the second decoction, add 5 to 6 times the amount of water and decoct for 1 to 2 hours. Combine the decoctions, filter, and concentrate the filtrate to a clear extract with a relative density of 1.16 to 1.22 (70℃). Decoct Pyrrosia lingua two to three times with water. For the first decoction, add 7 to 8 times the amount of water and decoct for 2 to 3 hours. Add 5-6 times the amount of water and decoct for 1-2 hours. Combine the decoctions, filter, and concentrate the filtrate to a clear paste with a relative density of 1.16-1.22 (70℃). Let it cool, add 1.5-2 times the volume of ethanol, stir well, and let it stand for 24-30 hours. Take the supernatant, recover the ethanol, concentrate it to an appropriate amount, mix it with the above clear paste, and continue to concentrate it to a clear paste with a relative density of 1.16-1.22 (70℃). Dry it, pulverize it, and you will get the product.

5. The compound Lysimachia christinae preparation according to claim 4, wherein the preparation method is as follows: Take four herbs: Lysimachia christinae, Plantago asiatica, Pyrrosia lingua, and corn silk. Decoct Lysimachia christinae, Plantago asiatica, and corn silk twice with water. For the first decoction, add 8 times the amount of water and decoct for 2 hours; for the second decoction, add 6 times the amount of water and decoct for 1 hour. Combine the decoctions, filter, and concentrate the filtrate to a clear extract with a relative density of 1.16–1.22 (70℃). Decoct Pyrrosia lingua twice with water. For the first decoction, add 8 times the amount of water and decoct for 2 hours; for the second decoction, add 6 times the amount of water and decoct for 1 hour. Combine the decoctions, filter, and concentrate the filtrate to a clear extract with a relative density of 1.16–1.22 (70℃). Cool, add 1.5 times the volume of ethanol, stir well, let stand for 24 hours, take the supernatant, recover the ethanol, concentrate to an appropriate volume, mix with the above clear extract, and continue to concentrate to a clear extract with a relative density of 1.16–1.22 (70℃). Dry, pulverize, and obtain the final product.

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