A traditional chinese medicine composition for treating stress urinary incontinence

Abstract

The application relates to the technical field of stress urinary incontinence treatment, and particularly discloses a traditional Chinese medicine composition for treating stress urinary incontinence, which is composed of the following raw medicinal materials in a weight ratio: 15g of raw astragalus, 15g of codonopsis, 15g of salvia miltiorrhiza, 10g of raspberry, 10g of immature bitter orange and 6g of bupleurum. The application is strictly designed according to a single-arm, single-center, clinical retrospective research, and an internationally commonly used urinary incontinence outcome index determination standard. The test result shows that, at the end of 4 weeks of treatment, 69 sample results show that the Qishizhiyi recipe combined with pelvic floor muscle training can significantly reduce the ICI-Q-SF score, the number of 24h urinary incontinence, and improve the I-QOL score, and the total effective rate reaches 89.86%, and there is no obvious toxic side effect. Through basic animal experiment research, it is shown that the Qishizhiyi recipe can significantly reduce collagen degradation, regulate hormone levels, reduce oxidative stress damage and inhibit cell apoptosis, and from the basic aspect, it is confirmed that the Qishizhiyi recipe has the effects of regulating connective tissue factors and hormone levels and inhibiting cell apoptosis.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of stress urinary incontinence, and more particularly to a traditional Chinese medicine composition for treating stress urinary incontinence. BACKGROUND

[0002] There are many epidemiological studies on female urinary incontinence, and different studies show that the prevalence of the disease varies greatly, which is related to the study population, measurement period and method for evaluating severity. Studies have found that 23%-45% of the female population have varying degrees of urinary incontinence, about 50% of which is SUI. The prevalence of SUI in Chinese women is about 18.9%, and the prevalence rate increases with age, reaching 28.2% at the age of 50-59, which is the peak age of SUI. The high incidence rate determines the high expenditure of the medical system in medical consultation and supply of urinary incontinence materials. However, due to the low social acceptance of this disease, many patients bear the economic burden of urinary incontinence pads themselves. A meta-analysis in 2004 assessed the social and economic burden of urinary incontinence, and the cumulative expenditure in Canada, Germany, Italy, Spain, Sweden and the United Kingdom was about 7 billion euros per year, and the expenditure in the United States in 2007 was 66 billion euros. China has entered an aging society, and urinary incontinence, an international and social disease positively correlated with age, will inevitably lead to more social and economic and health problems. Although many countries have developed standardized recommended guidelines, urinary incontinence has not yet been completely cured.

[0003] The theory of "spleen and kidney tonification" applied to the treatment of SUI is feasible: SUI belongs to the category of "bladder cough", "urinary incontinence" and "incontinence" in traditional Chinese medicine. Due to the weakness of the spleen in transportation, the bladder is malnourished and the opening and closing are out of order, or the muscles are malnourished and cannot hold; the kidney is weak in gasification, and the bladder is not conducive to opening and closing, resulting in incontinence. The spleen cannot transform the preheavenly qi, and the kidney cannot generate the postheavenly qi, eventually leading to deficiency of both the spleen and the kidney. The treatment is to "invigorate the spleen, tonify the kidney, tonify qi and activate blood, and shrink urine and stop leakage". In the formula, raw radix astragali and radix codonopsis are the monarch herbs for tonifying qi, among which raw radix astragali is sweet and warm, enters the spleen and lung meridians, and is a qi-tonifying herb in traditional Chinese medicine, which has the functions of tonifying qi and spleen, raising yang and reducing swelling, and astringing and promoting tissue regeneration. Radix codonopsis is sweet and flat, and is used with raw radix astragali. Rubus chingii is sweet and sour, flat, and can tonify the kidney, consolidate essence and shrink urine. Salvia miltiorrhiza activates blood and removes stasis, and Fructus Aurantii Immaturus breaks qi and removes stagnation, and Radix Bupleuri soothes the liver and promotes qi circulation, all of which are used as auxiliary herbs to make qi and blood flow, tonify qi without stagnation. The whole formula integrates the functions of tonifying the spleen, tonifying the kidney, activating blood and promoting qi circulation, and takes both the symptoms and the root into account, tonifying the spleen and the kidney. SUMMARY

[0004] In order to overcome the above-mentioned defects of the prior art, the present application provides a traditional Chinese medicine composition for treating stress urinary incontinence to solve the problems existing in the above background art.

[0005] The application provides the following technical scheme: a traditional Chinese medicine composition for treating stress urinary incontinence, which is composed of raw Huangqi 15g, Dangshen 15g, Danshen 15g, Moshire 10g, Jisheng 10g and Chaihu 6g by weight of raw materials; in the prescription, raw Huangqi and Dangshen are the monarch drugs for tonifying qi and benefiting the spleen, wherein raw Huangqi is sweet and warm, enters the spleen and lung channels, is a qi-tonifying drug in traditional Chinese medicine, has the effects of tonifying qi and the spleen, raising yang and lifting depression, and astringing and promoting tissue regeneration, Dangshen is sweet and flat, can invigorate the spleen and benefit qi, and is used with raw Huangqi; Moshire is sweet and sour, flat, can benefit the kidney, consolidate essence and reduce urine; Danshen can activate blood and remove stasis, Jisheng can break qi and remove stagnation, and Chaihu can soothe the liver and promote qi, and the three are the auxiliary drugs, so that qi is strong, blood flows, qi is supplemented without stagnation, the whole prescription integrates the spleen and kidney, activates blood, and promotes qi, and the prescription takes both the symptoms and the root cause into account, and supplements the spleen and the kidney;

[0006] Research status of Huangqi The main chemical components of Huangqi include flavonoids, saponins, polysaccharides and the like, and Huangqi has the effects of improving immune function, resisting oxidation, protecting heart and blood vessels, resisting tumors, promoting metabolism of the body, protecting liver, reducing blood sugar and the like.

[0007] Research status of Dangshen The main chemical components of Dangshen include alkynes, alkaloids, triterpenes and saponins, phenylpropanoids, lignans and flavonoids, and Dangshen has the effects of enhancing immune function of the body, protecting blood vessels, reducing blood sugar, nerve protection, resisting oxidation and resisting fatigue.

[0008] Research status of Moshire The main chemical components of Moshire include polysaccharides, flavonoids and phenols, terpenoids, alkaloids and organic acids, and Moshire has the effects of resisting oxidation, resisting aging, resisting inflammation and resisting tumors. It has been proved that Moshire can inhibit the expression of MMPs and inhibit the degradation of collagen.

[0009] Research status of Danshen The main chemical components of Danshen include tanshinones, salvianolic acids, volatile oils, polysaccharides and nitrogen-containing compounds, and Danshen can dilate blood vessels, improve blood flow and blood high coagulation state, and has the effects of resisting blood coagulation, resisting inflammation, resisting oxidation, resisting fibrosis, resisting tumors and protecting viscera.

[0010] Research status of Chaihu The main chemical components of Chaihu include Chaihu saponins, volatile oils, flavonoids and polysaccharides, and Chaihu has the pharmacological effects of resisting depression, resisting inflammation, immune regulation, protecting liver and the like.

[0011] Research status of Jisheng After being roasted with bran, Jisheng can be milder, so as to avoid damaging healthy qi; the main chemical components of Jisheng roasted with bran include flavonoids, alkaloids and volatile oils, and Jisheng has the effects of regulating the gastrointestinal tract, resisting inflammation, protecting liver and resisting ulcer.

[0012] Further, the traditional Chinese medicine composition needs to be soaked and decocted in the process of decoction.

[0013] Further, the soaking step comprises ① first soaking the medicine, soaking with cold water, and the soaking time is 30 to 50 minutes; ② the water added should be 7-12 times the weight of the decoction pieces, or the water added is soaked to 2-5 cm of the medicine surface, or the water added is calculated according to the expected liquid amount, the water absorption amount of the first decoction and the loss of the medicine liquid in the first decoction process, ③ the soaking liquid after soaking should not be discarded and should be put into the first decoction together with the soaked medicine.

[0014] Further, the decocting step comprises ① in the first decoction, first heat with strong fire to reach boiling, then change to slow decocting with weak fire and keep slight boiling, and decoct for 30-35 minutes; ② after the first decoction is completed, filter the medicine liquid with filter cloth or filter screen; after the medicine liquid of single dose decoction is filtered, wait for use; after the medicine liquid of multiple dose decoction is filtered, the medicine liquid should be treated with heat preservation; ③ the water added in the second decoction should be less than that in the first decoction, or the water added is calculated according to the expected liquid amount, the water absorption amount of the second decoction and the loss of the medicine liquid in the second decoction process; first heat with strong fire to reach boiling, then change to slow decocting with weak fire and keep slight boiling decocting to the specified time; generally, the decocting time of the second decoction should be slightly shorter than that of the first decoction, and the decocting time of the second decoction is 20-25 minutes; ④ when decocting, stir and prevent the medicine liquid from overflowing; control the decocting time and the fire, and prevent dry pot and burnt medicine; ⑤ after the second decoction is completed, filter the medicine liquid with filter cloth or filter screen meeting the food or medicine requirements; mix the medicine liquid of the second decoction with the medicine liquid of the first decoction when decocting single dose; mix the medicine liquid of the second decoction with the medicine liquid of the first decoction when decocting multiple dose, and make the mixed medicine liquid boil before packaging.

[0015] Technical effects and advantages of the present application:

[0016] The present application strictly follows the clinical randomized controlled, observational research design, and the determination standard of the international commonly used urinary incontinence outcome indicators according to the "Urinary Incontinence Diagnosis and Treatment Guide" (2022, "Chinese Urology and Male Disease Diagnosis and Treatment Guide"). The test results show that at the end of 4 weeks of treatment, 69 sample results show that Qishizhiyi Decoction combined with pelvic floor muscle training significantly improves the ICI-Q-SF score, reduces the number of 24h urinary incontinence, and improves the I-QOL score. The clinical total effective rate is 89.86%, and there is no obvious toxic and side effects.

[0017] The animal basic experiment research of the present application shows that Qishizhiyi Decoction can significantly reduce collagen degradation, regulate hormone levels, reduce oxidative stress damage and inhibit cell apoptosis, which confirms from the basic aspect that Qishizhiyi Decoction has the effects of regulating connective tissue factors and hormone levels and inhibiting cell apoptosis. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 Comparison of ALLP values of each group before and after intervention;

[0019] Figure 2HE staining of levator ani muscle tissue of rats in each group (x40);

[0020] Figure 3 Effect of Qishi Zhiyi Decoction on the injury of anterior vaginal wall of SUI rats (HE, 40x);

[0021] Figure 4 Effect of Qishi Zhiyi Decoction on the injury of anterior vaginal wall of SUI rats (Masson, 40x);

[0022] Figure 5 Effect of Qishi Zhiyi Decoction on the cell proliferation and apoptosis of anterior vaginal wall of SUI rats (TUNEL, 100x);

[0023] Figure 6 Concentration of serum ET-1 and NO of rats in each group after intervention;

[0024] Figure 7 Concentration of serum ROS and GSH-PX of rats in each group after intervention;

[0025] Figure 8 Concentration of serum E2 and T of rats in each group after intervention;

[0026] Figure 9 Expression level of related proteins of levator ani muscle tissue of rats in each group (x±s, n=4);

[0027] Figure 10 Expression level of related proteins of anterior vaginal wall of rats in each group (n=3). DETAILED DESCRIPTION

[0028] The technical solutions in the present application will be described clearly and completely in combination with the drawings in the present application, and additionally, the forms of each structure described in the following embodiments are only examples, and the Chinese medicine composition for treating stress urinary incontinence involved in the present application is not limited to each structure described in the following embodiments, and all other embodiments obtained by those skilled in the art without making creative efforts belong to the protection scope of the present application.

[0029] The application provides a traditional Chinese medicine composition for treating stress urinary incontinence, which is composed of raw Huangqi 15g, Dangshen 15g, Danshen 15g, Moshire 10g, Cishu 10g and Chaihu 6g by weight.

[0030] The main chemical components of Huangqi include flavones, saponins, polysaccharides and the like, and Huangqi has the effects of improving immune function, resisting oxidation, protecting heart and blood vessels, resisting tumor, promoting metabolism of the body, protecting liver and reducing blood sugar.

[0031] The main chemical components of Dangshen include alkynes, alkaloids, triterpenes and saponins, phenylpropanoids, lignans and flavones, and Dangshen has the effects of enhancing immune function of the body, protecting blood vessels, reducing blood sugar, protecting nerves, resisting oxidation and resisting fatigue.

[0032] The main chemical components of Moshire include polysaccharides, flavones and phenols, terpenoids, alkaloids and organic acids, and Moshire has the effects of resisting oxidation, resisting aging, resisting inflammation and resisting tumor. It has been proved that Moshire can inhibit expression of MMPs and inhibit degradation of collagen.

[0033] The main chemical components of Danshen include tanshinones, salvianolic acids, volatile oils, polysaccharides and nitrogen-containing compounds, and Danshen can dilate blood vessels, improve blood flow and blood high coagulation state, and has the effects of resisting blood coagulation, resisting inflammation, resisting oxidation, resisting fibrosis, resisting tumor and protecting viscera.

[0034] The main chemical components of Chaihu include Chaihu saponins, volatile oils, flavones and polysaccharides, and Chaihu has the pharmacological effects of resisting depression, resisting inflammation, regulating immunity, protecting liver and the like.

[0035] It is considered by medical experts in the past dynasties that Cishu can be roasted with bran to moderate the severe nature of Cishu so as to avoid damaging healthy qi. The main chemical components of bran-roasted Cishu include flavones, alkaloids and volatile oils, and the pharmacological effects of Cishu are shown in regulating gastrointestinal tract, resisting inflammation, protecting liver and resisting ulcer.

[0036] Further, the traditional Chinese medicine composition needs to be soaked and decocted in two steps during decoction.

[0037] Further, the soaking step includes ① first soaking the medicine with cold water, and the soaking time is thirty to fifty minutes; ② the water added should be 7-12 times the weight of the decoction pieces, or the water added should be 2-5 centimeters higher than the medicine surface, or the water added should be calculated according to the expected liquid amount, the water absorption amount of the first decoction, and the loss of the medicine liquid during the first decoction process; ③ the soaking liquid should not be discarded and should be put into the first decoction together with the soaked medicine.

[0038] Further, the decocting step includes ① during the first decoction, first heat with strong fire to reach boiling, then change to slow decoction with weak fire and keep slight boiling, and decoct for 30-35 minutes; ② after the first decoction is completed, filter the medicine liquid with filter cloth or filter screen; after the medicine liquid of single-dose decoction is filtered, wait for use; after the medicine liquid of multiple-dose decoction is filtered, also perform heat preservation; ③ the water added for the second decoction should be less than that for the first decoction, or the water added should be calculated according to the expected liquid amount, the water absorption amount of the second decoction, and the loss of the medicine liquid during the second decoction process; first heat with strong fire to reach boiling, then change to slow decoction with weak fire and keep slight boiling decoction to the specified time; generally, the decoction time of the second decoction should be slightly shorter than that of the first decoction, and the decoction time of the second decoction is 20-25 minutes; ④ when decocting, stir and prevent the medicine liquid from overflowing; control the decoction time and the fire, and prevent dry pot and burnt medicine; ⑤ after the second decoction is completed, filter the medicine liquid with filter cloth or filter screen meeting the food or medicine requirements; mix the medicine liquid of the second decoction with that of the first decoction when decocting single dose; mix the medicine liquid of the second decoction with that of the first decoction when decocting multiple doses, and make the mixed medicine liquid boil before packaging. Embodiment

[0039] Select 69 cases of female patients with mild to moderate stress urinary incontinence of spleen-kidney deficiency type diagnosed in Xiyuan Hospital of China Academy of Chinese Medical Sciences from January 2021 to December 2023.

[0040] Table 1 General information of patients

[0041] Patient baseline information n=69 Age / years 63（22~92） Course of disease / year 8.62（0.24~35） Parity / time 2（0~5） Classification / Example (%) Mild 14（24.64） Moderate 55（75.36）

[0042] The inclusion criteria are:

[0043] (1) Western medicine diagnosis according to the diagnosis and treatment guidelines for urinary surgery in China (2022 edition) stress urinary incontinence diagnosis standard;

[0044] (2) TCM syndrome differentiation type according to the diagnosis and treatment of Chinese medicine syndromes and the clinical research guidelines for new drugs of traditional Chinese medicine to develop the spleen-kidney deficiency syndrome standard: incontinence, cold limbs, soreness of the waist and knees, poor appetite, abdominal distension, abdominal distension, loose stool, fatigue, loose stool, soreness of the waist, tinnitus, pale tongue, and weak pulse.

[0045] (3) Complete clinical data, no missing items;

[0046] (4) Patients with stress urinary incontinence who have no contraindications for the drug used in this study.

[0047] Exclusion criteria are:

[0048] (1) Patients with severe urinary tract infection and other types of urinary incontinence;

[0049] (2) Patients with severe urinary incontinence caused by urethral sphincter atresia, ectopic ureter, etc. requiring surgical treatment;

[0050] (3) Vaginal bleeding or inflammation;

[0051] (4) Patients with severe systemic diseases;

[0052] (5) Patients with mental illness or language barriers;

[0053] (6) Pregnancy;

[0054] (7) Patients allergic to the drug;

[0055] (8) Patients without complete follow-up data before and after treatment or those who do not complete the treatment period.

[0056] Intervention measures are:

[0057] Qishizhiyi Decoction combined with pelvic floor muscle training. Qishizhiyi Decoction is composed of Huangqi 15g, Dangshen 15g, Danshen 15g, Moshire 10g, Cishu 10g, Chaihu 6g. One dose per day, decocted with water in the morning and evening, taken after meals, 100ml each time.

[0058] Course of treatment: 4 weeks of treatment, and no other similar drugs during treatment;

[0059] Observation indicators are:

[0060] ICI-Q-SF score

[0061] ICI-Q-SF questionnaires were collected before and after treatment, including the frequency of urine leakage, the amount of urine leakage, and the degree of impact on the quality of life. The total score is 21, with a minimum of 0. According to the International Continence Society's recommendations, the severity of urinary incontinence can be divided into three grades according to the score of the questionnaire: ≤7 points for mild; 8-13 points for moderate; 14-21 points for severe;

[0062] I-QOL score

[0063] I-QOL score was collected before and after treatment, including the psychological impact of urinary incontinence on patients, social barriers and behavioral restrictions, a total of 22 questions, each question has 5 grades, and each grade has different scores, from 1 to 5, and the lower the total score, the worse the quality of life of the patient with urinary incontinence.

[0064] 24h urine incontinence times

[0065] The number of 24h urine incontinence times of patients before and after treatment was compared;

[0066] Efficacy evaluation criteria

[0067] According to the Chinese "Female SUI Diagnosis and Treatment Guidelines (2017)", the efficacy was judged according to the improvement of symptoms and objective monitoring items. ① Ineffective: the clinical symptoms and urine incontinence times of the patient did not change significantly, and the urine pad test was positive; ② Effective: the number of urine leakage and symptoms of the patient were reduced compared with before treatment; ③ Marked effect: the number of urine leakage of the patient was reduced, the symptoms were reduced, and the number of urine pad use was significantly reduced; ④ Cure: the patient's urine leakage symptoms disappeared, no urine pad was used, and no urine leakage occurred. The total effective rate = (cured cases + effective cases + effective cases) / total cases x 100%;

[0068] Adverse reactions

[0069] According to the CTCAE adverse reaction evaluation standard, all adverse reactions and severity of patients during treatment were recorded.

[0070] Statistical methods are:

[0071] Statistical software SPSS 21.0 was used to process the data. The baseline data of patients were only descriptive analysis. Continuous variables conforming to normal distribution were expressed by mean ± standard deviation (xˉ±s), and those not conforming to normal distribution were expressed by median and upper and lower quartiles [M (P25, P75)]; The frequency and percentage of categorical variables were used. Continuous variables conforming to normal distribution were used for paired sample t test, and those not conforming to normal distribution were used for Wilcoxon signed rank test; McNemar test or McNemar-Bowker test was used for categorical variables. P<0.05 indicates that the difference is statistically significant.

[0072] Results

[0073] Efficacy analysis

[0074] The ICI-Q-SF score [5.30 (4.00, 7.00)] was significantly lower after 4 weeks of treatment than before treatment [9.21 (7.50, 11.00)], and the difference was statistically significant (P<0.01); the I-QOL score (64.34±4.79) was higher than before treatment (52.55±4.05), and the difference was statistically significant (P<0.01); the number of incontinence episodes in 24 hours [2.16 (1.00, 3.00)] was significantly reduced than before treatment [4.30 (3.00, 5.00)] (P<0.01).

[0075] Table 2 Comparison of scale scores and incontinence episodes before and after treatment

[0076] The total effective rate of clinical treatment was 89.86%.

[0077] Table 3 Efficacy determination criteria after treatment

[0078] Adverse reactions

[0079] Only 1 case of mild diarrhea occurred during treatment.

[0080] Conclusion

[0081] 1. Qishizhiyi Decoction combined with pelvic floor muscle training can improve the ICI-Q-SF score of patients with mild to moderate female SUI of spleen and kidney deficiency type;

[0082] 2. Qishizhiyi Decoction combined with pelvic floor muscle training can reduce the I-QOL score of patients with mild to moderate female SUI of spleen and kidney deficiency type;

[0083] 3. Qishizhiyi Decoction combined with pelvic floor muscle training can reduce the number of incontinence episodes in 24 hours in patients with mild to moderate female SUI of spleen and kidney deficiency type;

[0084] 4. Qishizhiyi Decoction combined with pelvic floor muscle training can improve the clinical cure rate and total effective rate of patients with mild to moderate female SUI of spleen and kidney deficiency type;

[0085] 5. Qishizhiyi Decoction combined with pelvic floor muscle training is safe for the treatment of patients with mild to moderate female SUI of spleen and kidney deficiency type. Example

[0086] Animal grouping and administration method

[0087] After 1 week of adaptive feeding, 60 SD female rats were randomly selected for 8 as blank control (Control), SUI rat model was prepared by "ovariectomy operation + vaginal dilatation method" combined modeling method, and the modeling was detected by urodynamics. The rats who met the standard were randomly divided into model group (Model), western medicine group (Midodrine), low dose of Qishiguan optimization group (QSKL-L), and high dose of Qishiguan optimization group (QSKL-H), 10 rats in each group.

[0088] The dose was converted according to the clinical dose and the animal body surface coefficient. Method: according to the body weight to calculate the dose, the blank group and the model group were given 0.1 ml·kg-1·d-1 of normal saline by gavage; the western medicine group was given 0.45 mg·kg-1·d-1 of midodrine hydrochloride solution, and the low dose group of Qishiguan optimization (equivalent dose) and the high dose group were given 6.39 g·kg-1·d-1 and 12.87 g·kg-1·d-1 of Qishiguan optimization, respectively. The intervention was performed by gavage at a fixed time every day, and the cycle was 2 weeks.

[0089] Sampling

[0090] After the last administration, the urodynamics was detected first, then the rats were deprived of water for 12 hours, weighed, and the abdominal cavity was opened along the abdominal midline after anesthesia. A horizontal incision was made on both sides of the bottom, and blood was taken from the abdominal aorta. After standing, centrifugation was performed under the following conditions: 4℃ environment, 3000 rpm, 15 min. The serum was first stored in a-20℃ refrigerator and then transferred to a-80℃ refrigerator for detection of sex hormones. The pubic symphysis was cut with scissors, and the cut pubic symphysis was separated and cut on both sides to fully expose the anterior wall of the vagina. The anterior wall of the vagina was cut along the external orifice of the vagina. The rat's perineum was separated, and the longitudinal muscle bundle running between the pubic symphysis and the coccyx, which was the pubococcygeal muscle in the levator ani muscle, was visible. The pubococcygeal muscle specimen was gently fixed with forceps, and 100 mg of the pubococcygeal muscle specimen was washed with normal saline. After washing, the specimen was fixed with formalin. The other side of the pubococcygeal muscle was cut in the same way, the tissue was wrapped in tin paper, and the tissue was quickly frozen in liquid nitrogen and stored in a-80℃ refrigerator.

[0091] Detection index

[0092] HE staining of levator ani muscle and anterior vaginal wall tissue

[0093] The specimen was fixed in 4% paraformaldehyde solution for 24 hours. After the specimen was taken out, gradient ethanol was used for dehydration treatment. Xylene was used for transparent treatment. After paraffin immersion, embedding, sectioning, deparaffinization, and hydration, hematoxylin and eosin staining was performed, and then the specimen was mounted. The pathological sections were scanned, and the histopathological changes were observed by SlideViewer software.

[0094] Masson staining of anterior vaginal wall

[0095] The vaginal anterior wall tissue section is routinely deparaffinized, hydrated, and then dyed in Weigert iron hematoxylin staining solution for 8 min; acidified ethanol differentiation solution is differentiated for 10 s, washed with water; Masson blue solution is returned to blue for 5 min, washed with water; ponceau red staining solution is dyed for 10 min; and weak acid working solution (distilled water: weak acid solution = 2:1) is washed for 1 min. Phosphomolybdate solution is washed for 2 min; weak acid working solution is washed for 1 min; and the solution is dyed in aniline blue staining solution for 2 min. Weak acid working solution is washed for 1 min; gradient ethanol is dehydrated, xylene is transparent, and after drying, neutral gum is sealed, and the pathological changes of the vaginal anterior wall tissue are observed under a microscope;

[0096] TUNEL staining of the vaginal anterior wall

[0097] The vaginal anterior wall paraffin section is deparaffinized in xylene, hydrated by gradient alcohol, 50 μL of reaction solution is added to the tissue, 37°C dark reaction is performed for 1 h; then 50 μL of Streptavidin-TRITC labeling solution is added, and the reaction is continued for 30 min; DAPI staining solution is dyed for 10 min, the section is washed with PBS buffer, and anti-fluorescence quencher is sealed, and the apoptosis of the vaginal anterior wall cells is observed under a fluorescence microscope;

[0098] Serum index detection

[0099] (1) Determination of vasoactive substance index: Endothelin-1 (ET-1): The ET-1 level is determined by double antibody sandwich ELISA according to the reagent kit instructions; Nitric oxide (NO) content: The NO level is determined by nitrate reductase method according to the reagent kit instructions.

[0100] (2) Determination of oxidative stress index: Reactive oxygen species (ROS) detection: The ROS level is determined by chemical fluorescence method according to the reagent kit instructions; Glutathione peroxidase (GSH-PX) detection: The GSH-PX level is determined by colorimetry according to the reagent kit instructions.

[0101] (3) Determination of serum hormone index: Testosterone (T) detection, estrogen (E2) detection: The T and E2 levels are determined by enzyme-linked immunoassay according to the reagent kit instructions;

[0102] Western-blot is used to detect the expression of MMP-9, TIMP-1, TGF-β1 protein in the levator ani muscle tissue, and the expression of pPI3K-P85, pAKT, BCL-2, BAX protein in the vaginal anterior wall tissue.

[0103] The levator ani muscle / vaginal anterior wall tissue in liquid nitrogen was taken out, lysed and centrifuged, total protein was collected after collecting vaginal anterior wall cells, BCA method was used for protein quantification, and denaturation was added after adding loading buffer. According to the molecular weight of the target protein, the separation gel concentration is 12%, 8%, and the concentration gel concentration is 5%. Take 20ug protein, gel electrophoresis, transfer membrane, completely immerse the membrane in 3% BSA-TBST at room temperature for 30min; after removing the blocking solution, dilute the primary antibody with 3% BSA-TBST, incubate at 4℃ overnight. After incubation at room temperature for 30min the next day, wash with TBST for 5 times, 3min / time. Dilute the secondary antibody with 5% skimmed milk powder-TBST, shake at room temperature for 40min, wash with TBST for 6 times, 3min / time. After ECL is added to the membrane, react for 3-5min, expose the film, develop for 2min, and fix. Use β-actin as an internal reference for correction. Scan the film and use ImageJ software to analyze the gray scale of the scanned bands.

[0104] II. Results

[0105] Qishi Zhiyi Decoction can significantly increase the ALPP value of SUI model rats, and improve the pathological morphology of levator ani muscle and vaginal anterior wall.

[0106] Comparison of ALLP values before and after intervention

[0107] Before intervention, the ALPP values of the model group, low-dose group, high-dose group and western medicine group were lower than those of the blank group (P<0.01); there was no difference between the model group, low-dose group, high-dose group and western medicine group (P>0.05).

[0108] After intervention, the ALPP values of each group increased; compared with the blank group, the ALPP value of the model group increased (P<0.01); compared with the model group, the ALPP values of the low-dose group and the high-dose group increased (P<0.05, P<0.01); the ALPP value of the western medicine group increased, and the difference was not statistically significant (P>0.05).

[0109] Before and after intervention, there was no significant change in the blank group (P>0.05); compared with before intervention, the ALPP value of the model group increased after intervention (P<0.05); the ALPP values of the low-dose group, the high-dose group and the western medicine group increased (P<0.01). The results are shown in Table 1 and Figure 1 ;

[0110] Table 1 Comparison of ALLP values of rats in each group before and after intervention (cmH2O, `c±s)

[0111] Group Number of cases (n) Before intervention After intervention t value P value Control 8 66.13±11.46 65.50±8.28 0.417 0.689 Model 10 29.80 ± 7.28 ** ]] 36.80 ± 3.26 ** ]] -2.837 0.020 Midodrine 10 28.70 ± 6.98 ** ]] 40.70±8.38 -4.37 0.002 QSKL-L 10 30.20 ± 10.38 ** ]] 46.00 ± 10.03 # ]] -4.296 0.002 QSKL-H 10 25.60 ± 4.99 ** ]] 57.80 ± 8.92 ## ]] -12.56 ＜0.001

[0112] Note: compared with the blank group, * P<0.05, ** P<0.01; compared with the model group, #P < 0.05 ## P < 0.01

[0113] Note: Compared with the blank group, * P < 0.05 ** P < 0.01; compared with the model group, # P < 0.05 ## P < 0.01; within the group, before and after comparison, compared with before intervention, ▲ P < 0.05 ▲▲ P < 0.01

[0114] HE staining of rat levator ani muscle tissue

[0115] HE staining results showed that in the blank group, the muscle fibers of the levator ani muscle of female SD rats were neatly and tightly arranged, continuous and intact, with uniform cytoplasmic staining and intercellular spaces. In the model group, the muscle fiber structure of the levator ani muscle was disordered, with breaks, reduced muscle cell content, uneven cytoplasmic staining, and inconsistent intercellular space sizes. After intervention, the muscle fiber arrangement of the levator ani muscle of rats was more neat and continuous than that of the model group, and the number of muscle fibers increased. See details... Figure 2 Note: A: Blank group; B: Model group; C: Western medicine group; D: Low-dose group of Qishi Zhiyi formula; E: High-dose group of Qishi Zhiyi formula.

[0116] HE staining of rat anterior vaginal wall tissue

[0117] In the control group, the smooth muscle bundles of the anterior vaginal wall of rats were thick and woven-like; in the model group, the smooth muscle bundles of the anterior vaginal wall were thin and disorganized, with thinner bundles and wider inter-bundle spaces. The pathological damage in the Western medicine group, the low-dose Qishi Zhiyi formula group, and the high-dose Qishi Zhiyi formula group showed varying degrees of reduction, manifested as partial restoration of normal smooth muscle cell structure and a decrease in the number of necrotic cells. Results are shown below. Figure 3 Note: A. Blank group; B. Model group; C. Western medicine group; D. Low-dose group of Qishi Zhiyi formula; E. High-dose group of Qishi Zhiyi formula.

[0118] MASSON staining

[0119] In the blank control group, the smooth muscle bundles of the anterior vaginal wall of rats were thick and woven, with abundant and dense collagen content, and fewer broken and disordered collagen. In the model group, the smooth muscle bundles of the anterior vaginal wall were thin and randomly distributed, with thinner bundles, wider inter-bundle spaces, and visible breaks in the muscle fibers. The vascular and nerve tissue components were also reduced. The pathological damage in the Western medicine group, the low-dose Qishi Zhiyi formula group, and the high-dose Qishi Zhiyi formula group showed varying degrees of reduction, manifested as partial restoration of normal smooth muscle cell structure, thicker muscle bundles, and denser muscle fibers. Masson staining results are shown below. Figure 4 Note: A. Blank group; B. Model group; C. Western medicine group; D. Low-dose group of Qishi Zhiyi formula; E. High-dose group of Qishi Zhiyi formula.

[0120] TUNEL staining

[0121] Compared with the blank group, the number of TUNEL positive cells in the SUI model group was significantly increased (P<0.01); after intervention, compared with the model group, the number of TUNEL positive cells in the western medicine group, the high dose of Qishizhiyi Decoction group and the low dose of Qishizhiyi Decoction group was significantly reduced (P<0.01). See Table 2 and Figure 5 Note: A blank group; B model group; C western medicine group; D low dose of Qishizhiyi Decoction group; E high dose of Qishizhiyi Decoction.

[0122] Table 2 Comparison of vaginal anterior wall cell apoptosis rate of rats in each group (x ± s)

[0123] Note: Compared with the blank group, * P<0.05, ** P<0.01; compared with the model group, # P<0.05, ## P<0.01

[0124] Effect of Qishizhiyi Decoction on serum indexes of rats

[0125] Effect of Qishizhiyi Decoction on vascular endothelial active substances

[0126] Compared with the blank group, the serum ET-1 concentration of rats in the SUI model group was significantly increased, and the NO concentration was significantly reduced (P<0.01); compared with the model group, the serum ET-1 concentration of rats in the low dose group, the high dose group and the western medicine group was significantly reduced (P<0.01); the serum NO concentration of rats in the high dose group and the western medicine group was significantly increased (P<0.01); see Table 3 and Figure 6 Note: Compared with the blank group, * P<0.05, ** P<0.01; compared with the model group, # P<0.05, ## P<0.01.

[0127] Table 3 Serum ET-1 and NO concentrations of rats in each group after intervention (x ± s)

[0128] Group Number n ET-1 (pg / ml) NO (umol / L) Control 8 56.11±2.32 49.94±3.87 Model 10 85.10 ± 9.44 ** ]] 30.01 ± 6.48 ** ]] Midodrine 10 65.01 ± 4.99 ## ]] 41.98 ± 8.44 ## ]] QSKL-L 10 73.37 ± 4.43 ## ]] 28.96±5.15 QSKL-H 10 61.25 ± 7.34 ## ]] 44.84 ± 3.12 ## ]]

[0129] Note: Compared with the blank group, * P<0.05, ** P<0.01; compared with the model group, # P<0.05, ## P<0.01

[0130] Effect of Qishizhiyi Decoction on oxidative stress

[0131] Compared with the blank group, the ROS concentration of the SUI model group increased, and the GSH-PX concentration decreased (P<0.01); after the intervention, compared with the model group, the ROS concentration of the low-dose group, the high-dose group, and the western medicine group decreased (P<0.01); the GSH-PX concentration increased (P<0.01), and the results are shown in Table 4 and Figure 7 Note: Compared with the blank group, * P<0.05, ** P<0.01; compared with the model group, # P<0.05, ## P<0.01.

[0132] Table 4 Serum ROS and GSH-PX concentrations of rats in each group after intervention (x±s)

[0133] Group Number n ROS (fluorescence intensity / ml) GSH-PX (U / ml) Control 8 238.67±12.63 479.50±16.72 Model 10 734.77 ± 117.32 ** ]] 278.63 ± 28.48 ** ]] Midodrine 10 284.21 ± 31.52 ## ]] 432.91 ± 28.28 ## ]] QSKL-L 10 428.42 ± 47.80 ## ]] 384.68 ± 34.86 ## ]] QSKL-H 10 272.58 ± 42.05 ## ]] 451.08 ± 24.64 ## ]]

[0134] Note: Compared with the blank group, *P<0.05, **P<0.01; compared with the model group, #P<0.05, ##P<0.01

[0135] Effect of Qishizhi Decoction on serum hormone levels

[0136] Compared with the blank group, the E2 and T concentrations of the SUI model group decreased (P<0.01); after the intervention, compared with the model group, the E2 concentration of the low-dose group, the high-dose group, and the western medicine group increased (P<0.01); the T concentration of the high-dose group increased (P<0.01), and the results are shown in Table 5 and Figure 8 Note: Compared with the blank group, * P<0.05, ** P<0.01; compared with the model group, # P<0.05, ## P<0.01.

[0137] Table 5 Serum E2 and T concentrations of rats in each group after intervention (x±s)

[0138] Group Number n E2 (pg / ml) T (ng / ml) Control 8 34.65±5.74 0.68±0.08 Model 10 15.54 ± 2.55 ** ]] 0.45 ± 0.16 ** ]] Midodrine 10 28.62 ± 3.33 ## ]] 0.54±0.20 QSKL-L 10 28.53 ± 3.97 ## ]] 0.54±0.10 QSKL-H 10 31.07 ± 4.76 ## ]] 0.65 ± 0.10 ## ]]

[0139] Note: Compared with the blank group, * P<0.05, ** P<0.01; compared with the model group, # P<0.05, ## P<0.01

[0140] Protein expression

[0141] MMP-9, TIMP-1, and TGF-β1 protein expression in levator ani muscle tissue

[0142] Compared with the blank group, the expression of MMP-9, TIMP-1 and TGF-β1 proteins in the SUI model group was significantly increased (P<0.01); after intervention, the expression of MMP-9 protein in the low-dose group and the high-dose group was significantly lower than that in the model group (P<0.01, P<0.05); the expression of MMP-9 protein in the western medicine group was lower than that in the model group, but there was no significant difference (P>0.05); the expression of TIMP-1 protein was increased after intervention with western medicine and Qishi Zhiyi Decoction, but the difference was not statistically significant (P>0.05); the expression of TGF-β1 protein was reduced after intervention with western medicine and Qishi Zhiyi Decoction, but the difference was not statistically significant (P>0.05); see Table 6 and Figure 9 Note: Compared with the blank group, * P<0.05, ** P<0.01; compared with the model group, # P<0.05, ## P<0.01

[0143] Table 6 Protein expression in the levator ani muscle tissue of rats in each group after intervention (x±s)

[0144] Group Number n MMP-9 TIMP-1 TGF-β1 Control 4 0.30±0.06 0.16±0.09 0.29±0.09 Model 4 0.62 ± 0.21 ** ]] 0.40 ± 0.12 ** ]] 0.60 ± 0.10 ** ]] Midodrine 4 0.42±0.11 0.49±0.17 0.42±0.19 QSKL-L 4 0.33 ± 0.09 ## ]] 0.52±0.18 0.47±0.21 QSKL-H 4 0.34 ± 0.16 # ]] 0.52±0.15 0.50±0.19

[0145] Note: Compared with the blank group, * P<0.05, ** P<0.01; compared with the model group, # P<0.05, ## P<0.01

[0146] Effect on the expression of pPI3K-P85, pAKT, BCL-2 and BAX proteins in the anterior vaginal wall tissue

[0147] Compared with the blank group, the expression of p-PI3K and p-AKT proteins in the SUI model rats was significantly reduced (P<0.01, P<0.05), and there was no statistically significant difference in the expression of Bcl-2 protein (P>0.05), but the expression of Bax protein was significantly increased (P<0.01). After intervention, compared with the model group, the expression of p-PI3K and p-AKT proteins in the anterior vaginal wall of rats in the high-dose Qishi Zhiyi Decoction group was significantly increased (P<0.05, P<0.05), and there was no statistically significant difference in the other groups; there was no significant difference in the expression of Bcl-2 protein in the anterior vaginal wall of rats in each group (P>0.05); there was no significant difference in the expression of Bax protein in the anterior vaginal wall of rats in the western medicine group (P>0.05), but the expression of Bax protein was reduced in the high-dose and low-dose Qishi Zhiyi Decoction groups (P<0.01, P<0.05); see Figure 10 .

[0148] Conclusion

[0149] 1、Qishi Zhiyi Decoction can significantly increase the ALPP value of SUI model rats, improve the pathological morphology of levator ani muscle and anterior vaginal wall, and reduce cell apoptosis.

[0150] 2、Qishi Zhiyi Decoction can regulate the content of serum TE-1, NO, ROS, GSH-PX, E2, T and other related indicators in SUI rats, suggesting that Qishi Zhiyi Decoction may play a therapeutic role by improving pelvic microcirculation, reducing oxidative stress damage, and regulating hormone levels.

[0151] 3、Qishi Zhiyi Decoction may improve SUI by reducing the expression of MMP-9, reducing collagen degradation, and promoting TIMP / MMP balance.

[0152] 4、Qishi Zhiyi Decoction may improve SUI by increasing the expression of PI3K and AKT proteins, reducing the expression of Bax protein, and inhibiting apoptosis of anterior vaginal wall cells through the PI3K\AKT pathway.

[0153] Secondly, the drawings of the embodiments of the present application only involve the structures related to the embodiments of the present disclosure, and other structures can refer to the usual design, and in the case of no conflict, the same embodiment and different embodiments of the present application can be combined with each other;

[0154] Finally, the above only describes the preferred embodiments of the present application and is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A traditional Chinese medicine composition for treating stress urinary incontinence, characterized in that: It is composed of raw medicinal materials with the following weights: Radix Astragali 15g, Codonopsis 15g, Salvia 15g, Ribes 10g, Fructus Aurantii Immaturus 10g, Bupleurum 6g. ​ 2. The Chinese medicine composition for treating stress urinary incontinence according to claim 1, characterized in that: The traditional Chinese medicine composition needs to pass through two steps of soaking and decocting in the process of decocting.

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