Traditional Chinese medicine compound oral preparation for treating male oligoasthenospermia

Abstract

The invention discloses a traditional Chinese medicine compound oral preparation for treating male oligoasthenospermia, and belongs to the technical field of traditional Chinese medicine preparations. The rhizoma polygonati processed with wine, the prepared rehmannia root and the like are used for nourishing the liver and kidney to consolidate congenital, the codonopsis pilosula, the astragalus membranaceus and the like are used for invigorating spleen and replenishing qi to complement postnatal, the fried cowherb seed is used for activating blood circulation, and the salted semen plantaginis is used for promoting diuresis, so that the problems of insufficient spermatogenesis and low vitality caused by deficiency of the spleen and the kidney are fundamentally solved. Experiments and clinical researches show that the preparation can significantly improve the sperm quantity, activity and activity rate of oligoasthenospermia model mice and patients, the clinical total effective rate reaches 90%, the pregnancy rate reaches 53.3%, and the curative effect is superior to that of Wuzi Yanzong pills in a control group. The action mechanism covers regulation of blood lipid metabolism, improvement of hypothalamus-pituitary-gonad axis functions, repair of testicular seminiferous tubule structural damage and reduction of lipid deposition, and meanwhile, oxidative stress and inflammatory injury are relieved and spermatogenic cell functions are protected by regulating and controlling a PPAR signal channel and inhibiting molecular pathways such as Cga / Sting mediated cell apoptosis.

Description

Technical Field

[0001] The present invention belongs to the technical field of traditional Chinese medicine preparations, and particularly relates to a compound oral preparation of traditional Chinese medicine for treating oligoasthenospermia in men. Background Art

[0002] Male infertility generally refers to a disease in which a couple of childbearing age cohabits for more than 1 year, has normal sexual life, does not take any contraceptive measures, the female is normal, and the female cannot conceive due to male factors, or although pregnancy occurs, it cannot be maintained until the fetus is normally delivered. According to domestic literature reports, the incidence of infertility in couples of childbearing age reaches 12.5%, and it shows an increasing trend year by year. Among infertility cases, those caused by male factors account for about 51.3%, and oligoasthenospermia is the main cause. With the acceleration of the modern social life rhythm, the low age of smoking and drinking, many bad living habits, and environmental pollution and other factors, the incidence of male infertility shows an increasing trend compared with the past, and the sperm quality shows a continuous downward trend compared with the past. According to a recent Meta-analysis, in the past 40 years, the average sperm count and sperm density of adult men globally have decreased by 60% and 52% respectively, and this downward trend continues. Oligoasthenospermia is the most common cause of male infertility, accounting for about 46% of the causes of male infertility, and its incidence is increasing year by year.

[0003] Oligoasthenospermia is the most common cause of male infertility, and its incidence is increasing year by year. Oligoasthenospermia includes oligospermia and asthenozoospermia (AZS). According to the standards of the 5th edition of the WHO "Laboratory Manual for the Examination and Processing of Human Semen", the total number of spermatozoa in one ejaculation < 39×10 6 / mL, or the sperm concentration < 15×10 6 / mL is diagnosed as oligospermia, and the percentage of spermatozoa with forward motility < 32% is diagnosed as asthenozoospermia. Since the two often coexist clinically, they are collectively referred to as oligoasthenospermia.

[0004] For the treatment of oligoasthenospermia, it mainly includes drug treatment and microsurgical treatment, etc., and their purposes are all to improve sperm quality so as to achieve pregnancy. Relevant clinical studies at home and abroad have shown that many drugs can better improve sperm quality. Since its pathogenesis has not been fully understood, the treatment effect of western medicine is not very satisfactory. And the emerging assisted reproductive technology cannot be accepted by most families due to its potential genetic risks. With the adjustment of China's population policy, there is an urgent need to explore safe and effective treatment methods at present.

[0005] There's no specific name for oligoasthenozoospermia in Traditional Chinese Medicine literature. Based on its clinical characteristics, the condition can be categorized as "infertility," "low sperm count," "thin sperm," or "clear sperm." The Huangdi Neijing (Huangdi Neijing) states that human reproductive function and growth and development are both external manifestations of the strength or weakness of kidney essence and qi. Only when kidney essence and qi are sufficient can the celestial essence flourish, the sperm chambers be filled, and the essence and qi overflow, allowing the union of yin and yang to produce children. If the kidney's function of sealing and storing essence declines, kidney essence deficiency can result in low sperm count, cold sperm, and infertility. The essence stored in the kidneys includes the innate essence inherited from parents and the acquired essence generated from the spleen and stomach. The Yili Zhenchuan states: "Without the innate essence, the acquired essence cannot stand; without the acquired essence, the innate essence cannot be born." The generation of acquired essence depends on the innate essence, and the abundance of innate essence relies on the nourishment of acquired essence. Kidney yang is the root of the body's yang energy, driving the generation of essence and blood and driving the production and maturation of sperm. If kidney yang is deficient, the energy required to transform reproductive essence into energy is insufficient. If reproductive essence is not warmed, sperm motility is weakened. The continuous process of "yang transforming into qi"—kidney yang warms spleen yang, and grain qi nourishes primordial qi—repeatedly nourishes and promotes the innate and acquired essence, driving the transformation of the human body's essence. This is the foundation of life. Li Dongyuan believed that insufficient spleen and stomach transformation is due to excess yin and insufficient yang. That is, the spleen and stomach's transformation of grain and water essence requires the impetus of spleen yang. If spleen yang is deficient, the spleen and stomach's function of transportation and transformation is impaired, and the transformation of acquired essence becomes inefficient. If spleen and kidney yang are deficient, the internal production of essence to support the innate is inadequate, and the external transformation of grain to nourish the body is inadequate.

[0006] Spleen deficiency disrupts the transport and transformation functions of the kidneys, depriving them of the energy needed to transform grain and water into fine essence. This hinders the distribution of vital essence and qi, preventing the kidneys from nourishing their innate essence. Kidney deficiency, a result of insufficient innate essence, also hinders the spleen's ability to warm the body. Combined spleen and kidney deficiency leads to a lack of kidney storage, impairing the production of reproductive essence and impuriting the stored essence, ultimately leading to infertility. In summary, it is widely recognized by physicians that combined spleen and kidney deficiency is the fundamental pathogenesis of oligoasthenospermia.

[0007] Existing Traditional Chinese Medicine (TCM) treatments are mostly based on the theory of "warming and tonifying the spleen and kidneys." For example, the classic formula Jujing Decoction has been reported to have an efficacy rate of up to 85.77%. However, current TCM treatments for oligoasthenospermia still have significant flaws: First, the mechanisms of action of most TCM compound formulas have not been fully validated through modern pharmacological experiments, and there is a lack of molecular biological research on the regulation of sperm production, maturation, and motility. Second, existing preparations lack formulation optimization and dosage standardization, and clinical efficacy evaluation relies on traditional experience. These lack support from large-scale, multicenter controlled trial data, making it difficult to meet the demands of modern medicine for precision medicine. Therefore, developing an oral TCM compound formulation based on TCM theory, with a clear mechanism of action and definite efficacy, has important clinical significance and social value. Summary of the Invention

[0008] In view of the above situation, to overcome the defects of the existing technology, based on the theory of "deficiency of both the spleen and kidney" in traditional Chinese medicine and combined with modern pharmacological research, the present invention provides a traditional Chinese medicine compound oral preparation for treating male oligoasthenospermia with a clear mechanism of action, definite curative effect and high safety.

[0009] To achieve the above object, the following technical solution is adopted: The present invention provides a traditional Chinese medicine compound oral preparation for treating male oligoasthenospermia, which is prepared from the following raw materials in parts by weight: 3-35 parts of wine-processed polygonatum sibiricum, 4-22 parts of prepared rehmannia root, 3-25 parts of cuscuta chinensis, 4-23 parts of codonopsis pilosula, 8-32 parts of astragalus membranaceus, 3-24 parts of poria cocos, 4-26 parts of wolfberry fruit, 2-22 parts of semen plantaginis (decocted in a wrapped form), 3-25 parts of dried tangerine peel, 3-18 parts of stir-fried atractylodes lancea, 2-24 parts of epimedium brevicornum, 3-23 parts of stir-fried semen vaccariae.

[0010] Among them, wine-processed polygonatum sibiricum: As the monarch drug, it has the effects of tonifying essence and marrow, replenishing qi and nourishing yin. After being processed with wine, it can better nourish the "foundation of prenatal and postnatal constitutions", nourish the prenatal to support the postnatal, nourish the postnatal to nourish the prenatal, enhance its tonifying effect, promote the generation of essence and blood, and provide a material basis for sperm production.

[0011] Prepared rehmannia root: An important drug for nourishing blood and yin, tonifying the kidney and filling essence. It can nourish kidney yin, fill essence and marrow, help promote sperm production, improve sperm quality, and is a commonly used drug for treating kidney deficiency and essence deficiency.

[0012] Cuscuta chinensis: Sweet in taste and warm in nature, belonging to the liver, kidney and spleen meridians. It can tonify the kidney and benefit essence, nourish the liver and improve eyesight, and arrest miscarriage and diarrhea. For oligoasthenospermia caused by insufficient kidney essence, it can enhance kidney function, improve sperm motility and quantity.

[0013] Codonopsis pilosula: It has the effects of invigorating the spleen and benefiting the lung, nourishing blood and promoting the production of body fluid. It can enhance the function of the spleen and stomach, promote the transportation and transformation of water and grain essence, provide sufficient nutrition for the body, and thus help sperm production and development, and improve sperm quality.

[0014] Astragalus membranaceus: Tonifying qi and lifting yang, consolidating the exterior and stopping sweating, promoting diuresis and relieving edema, promoting the production of body fluid and nourishing blood. When combined with codonopsis pilosula, it enhances the power of warming yang and tonifying qi, can improve sperm motility. At the same time, astragalus membranaceus combined with angelica sinensis forms Danggui Buxue Decoction, which can tonify qi and nourish blood, and provide power for sperm production and activity.

[0015] Poria cocos: Promoting diuresis to eliminate dampness, strengthening the spleen and calming the mind. When combined with semen plantaginis, it can clear and eliminate dampness from sperm, expel pathogenic dampness in the body, create a good internal environment for sperm growth, and ensure sperm purity.

[0016] Wolfberry fruit: Nourishing the liver and kidney, tonifying essence and improving eyesight. It is rich in various nutrients and active substances, can nourish the liver and kidney, supplement kidney essence, improve sperm quality and quantity, and enhance reproductive function.

[0017] Plantago asiatica L. semen praeparatum: clearing heat and promoting diuresis, inducing diuresis for treating stranguria, draining dampness and stopping diarrhea, improving eyesight, resolving phlegm. After being processed with salt, its effect of entering the kidney meridian is enhanced. Combined with Poria cocos, it clears and promotes the damp-heat in the lower energizer, can improve the internal environment of the reproductive system, and helps to improve sperm quality.

[0018] Citrus reticulata Blanco pericarpium: regulating qi and strengthening the spleen, drying dampness and resolving phlegm. Combined with Atractylodes lancea (Thunb.) DC. stir-fried, it can enhance the function of strengthening the spleen and drying dampness, enabling the spleen and stomach to function healthily, the damp pathogen to be transformed, and qi and blood to be generated smoothly, thus being beneficial to the generation and development of sperm. At the same time, it can make the tonic not stagnant.

[0019] Atractylodes lancea (Thunb.) DC. stir-fried: drying dampness and strengthening the spleen, dispelling wind and cold. It can improve the transportation and transformation function of the spleen and stomach, remove dampness in the body, provide good conditions for the generation and maturation of sperm, and cooperate with Citrus reticulata Blanco pericarpium to regulate the qi movement and water metabolism of the body.

[0020] Epimedii folium: tonifying the kidney yang, strengthening the muscles and bones, dispelling wind and dampness. It can warm and tonify the kidney yang, provide the driving force for the production and maturation of sperm, enhance the motility of sperm, improve male reproductive function, and is a commonly used drug for treating oligospermia and asthenospermia caused by deficiency of kidney yang.

[0021] Vaccaria segetalis (Neck.) Garcke stir-fried: promoting blood circulation to dredge the channels, inducing lactation and detumescence, inducing diuresis for treating stranguria. It can activate blood circulation and dredge collaterals, improve the blood circulation of the reproductive system, create good conditions for the generation and transportation of sperm, enable sperm to reach the fertilization site better, and increase the pregnancy rate.

[0022] Preferably, the oral traditional Chinese medicine compound preparation is made from the following raw materials in parts by weight: Polygonatum sibiricum Red. wine-processed 8 - 25 parts, Rehmannia glutinosa Libosch. preparata 8 - 18 parts, Cuscuta chinensis Lam. 8 - 18 parts, Codonopsis pilosula (Franch.) Nannf. 8 - 18 parts, Astragalus membranaceus (Fisch.) Bge. 12 - 28 parts, Poria cocos 8 - 18 parts, Lycium barbarum L. 8 - 18 parts, Plantago asiatica L. semen praeparatum (decocted in a wrapped manner) 6 - 18 parts, Citrus reticulata Blanco pericarpium 8 - 18 parts, Atractylodes lancea (Thunb.) DC. stir-fried 6 - 12 parts, Epimedii folium 6 - 18 parts, Vaccaria segetalis (Neck.) Garcke stir-fried 8 - 18 parts.

[0023] Preferably, the oral traditional Chinese medicine compound preparation is made from the following raw materials in parts by weight: Polygonatum sibiricum Red. wine-processed 10 - 22 parts, Rehmannia glutinosa Libosch. preparata 9 - 16 parts, Cuscuta chinensis Lam. 9 - 16 parts, Codonopsis pilosula (Franch.) Nannf. 9 - 16 parts, Astragalus membranaceus (Fisch.) Bge. 15 - 25 parts, Poria cocos 9 - 16 parts, Lycium barbarum L. 9 - 16 parts, Plantago asiatica L. semen praeparatum (decocted in a wrapped manner) 8 - 16 parts, Citrus reticulata Blanco pericarpium 9 - 16 parts, Atractylodes lancea (Thunb.) DC. stir-fried 7 - 10 parts, Epimedii folium 9 - 16 parts, Vaccaria segetalis (Neck.) Garcke stir-fried 9 - 16 parts.

[0024] Preferably, the oral traditional Chinese medicine compound preparation is made from the following raw materials in parts by weight: Polygonatum sibiricum Red. wine-processed 15 parts, Rehmannia glutinosa Libosch. preparata 10 parts, Cuscuta chinensis Lam. 10 parts, Codonopsis pilosula (Franch.) Nannf. 10 parts, Astragalus membranaceus (Fisch.) Bge. 20 parts, Poria cocos 10 parts, Lycium barbarum L. 10 parts, Plantago asiatica L. semen praeparatum (decocted in a wrapped manner) 10 parts, Citrus reticulata Blanco pericarpium 10 parts, Atractylodes lancea (Thunb.) DC. stir-fried 9 parts, Epimedii folium 10 parts, Vaccaria segetalis (Neck.) Garcke stir-fried 10 parts.

[0025] Most preferably, the oral traditional Chinese medicine compound preparation is made from the following raw materials: 15 g of polygonatum sibiricum redolent of wine, 10 g of rehmannia glutinosa, 10 g of cuscuta chinensis, 10 g of codonopsis pilosula, 20 g of astragalus membranaceus, 10 g of poria cocos, 10 g of wolfberry fruit, 10 g of plantago asiatica l. (decocted in a wrapped form), 10 g of dried tangerine peel, 9 g of stir-fried atractylodes lancea, 10 g of epimedium brevicornum, 10 g of stir-fried semen vaccariae.

[0026] Further, for the oral traditional Chinese medicine compound preparation, each time it is decocted with water, 200 mL of juice is taken, administered orally twice a day, 1 hour after meals, and 12 weeks is taken as one course of treatment, with a total of 1 course of treatment.

[0027] The beneficial effects of the present invention are as follows: By using polygonatum sibiricum redolent of wine, rehmannia glutinosa, etc. to nourish the liver and kidney to consolidate the congenital endowment, and codonopsis pilosula, astragalus membranaceus, etc. to strengthen the spleen and replenish qi to enrich the acquired endowment, and being compatibly formulated with stir-fried semen vaccariae for promoting blood circulation and semen plantago asiatica l. for promoting diuresis, the problem of insufficient sperm production and low motility caused by spleen and kidney deficiency is fundamentally solved. Experimental and clinical studies have shown that this preparation can significantly increase the sperm count, motility and motility rate of mice with oligoasthenospermia model and patients, with a total clinical effective rate of 90% and a pregnancy rate of 53.3%, and the curative effect is better than that of the control group's five-seed fertility pill; its mechanism of action covers regulating blood lipid metabolism (decreasing TC, TG, LDL-C, increasing HDL-C), improving the function of the hypothalamic-pituitary-gonadal axis (elevating testosterone level), repairing the structural damage of testicular seminiferous tubules and reducing lipid deposition, and at the same time, through molecular pathways such as regulating the PPAR signaling pathway and inhibiting Cgas / Sting-mediated apoptosis (downregulating Caspase3 and upregulating Bcl-2), reducing oxidative stress and inflammatory damage, and protecting the function of spermatogenic cells. Description of the Drawings

[0028] Figure 1 It is an image of the effect of polygonatum sibiricum yulin decoction of the present invention on the transcriptome of obese mice with oligoasthenospermia;

[0029] Figure 2 It is an image of the effect of polygonatum sibiricum yulin decoction of the present invention on the testicular function of obese mice with oligoasthenospermia;

[0030] Figure 3 It shows that polygonatum sibiricum yulin decoction can improve the structural damage of the testis in model mice. Representative pictures of HE and oil red O staining results of polygonatum sibiricum yulin decoction on the testis of mice

[0031] Figure 4 It is an image of the western blot results for detecting the protein expression levels of Cgas, Sting, Casepase3, Bcl-2 and Ppar-γ.

[0032] The drawings are used to provide further understanding of the present invention, and constitute a part of the specification, and are used to explain the present invention together with the embodiments of the present invention, and do not constitute a limitation to the present invention. Detailed Embodiments

[0033] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0034] Unless otherwise defined, all professional and scientific terms used herein have the same meaning as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to the described content can be applied to the present invention. The preferred implementation methods and materials described herein are only for illustrative purposes and do not limit the content of this application.

[0035] The experimental methods in the following embodiments are all conventional methods unless otherwise specified. The test materials used in the following embodiments are all obtained from commercial channels unless otherwise specified.

[0036] Example 1

[0037] A traditional Chinese medicine compound oral preparation for treating oligoasthenospermia in men (hereinafter referred to as Huangjing Yulin Decoction) is made from the following raw materials: 15 g of processed Huangjing with wine, 10 g of cooked Rehmannia root, 10 g of Cuscuta chinensis, 10 g of Codonopsis pilosula, 20 g of Astragalus membranaceus, 10 g of Poria cocos, 10 g of Lycium barbarum, 10 g of Plantago asiatica L. with salt (decocted in a package), 10 g of Citrus reticulata Blanco, 9 g of stir-fried Atractylodes lancea, 10 g of Epimedium brevicornu Maxim., and 10 g of stir-fried Vaccaria segetalis (Neck.) Garcke.

[0038] Test Example 1

[0039] Effect of Huangjing Yulin Decoction on spermatogenic function of obese oligoasthenospermia model mice

[0040] I. Materials and experimental methods:

[0041] (1) Materials and reagents:

[0042] The Huangjing Yulin Decoction prepared according to Example 1 (including processed polygonatum sibiricum, prepared rehmannia root, cuscuta chinensis, codonopsis pilosula, astragalus membranaceus, poria cocos, wolfberry fruit, semen plantaginis, tangerine peel, stir-fried atractylodes lancea, epimedium brevicornu, stir-fried semen vaccariae), among which stir-fried semen vaccariae, stir-fried atractylodes lancea, and astragalus membranaceus are purchased from Anhui Xiehe Pharmaceutical Slices Co., Ltd. The batch number of stir-fried semen vaccariae is: 23112817, the batch number of stir-fried atractylodes lancea is 23102608, and the batch number of astragalus membranaceus is: 23121520. Codonopsis pilosula, tangerine peel, epimedium brevicornu, semen plantaginis, wolfberry fruit, poria cocos, cuscuta chinensis, prepared rehmannia root, and processed polygonatum sibiricum are purchased from Bozhou Huqiao Pharmaceutical Co., Ltd. The batch number of codonopsis pilosula is: 24012401, the batch number of tangerine peel is: 2402180672, the batch number of epimedium brevicornu is: 2311270332, the batch number of semen plantaginis is: 2312280142, the batch number of wolfberry fruit is: 2401040032, the batch number of poria cocos is: 2401280013, the batch number of cuscuta chinensis is: 2311170092, the batch number of prepared rehmannia root is: 2401090162, and the batch number of processed polygonatum sibiricum is: 2306060082. The Huangjing Yulin Decoction is prepared into a decoction containing 840 mL of crude drug, sealed and stored in a refrigerator at 4 °C for standby.

[0043] The Wuzi Yanzong Pills are purchased from Tongrentang Pharmaceutical Co., Ltd., Beijing Tongrentang Co., Ltd.

[0044] The high-fat diet is purchased from Anhui Hefei Qingyuan Biotechnology Co., Ltd. The formula of the high-fat diet (mass fraction) is: lard 5%, sugar 5%, egg yolk powder 5%, cholesterol 1%, and basal maintenance diet 84%.

[0045] The normal diet feed (basal maintenance diet) is purchased from Beijing Huafukang Biotechnology Co., Ltd., and the batch number is 2210220909.

[0046] The triglyceride (TG) kit (batch number: 20230526), total cholesterol (T-CHO) kit (batch number: 20230525), high-density lipoprotein cholesterol (HDL-C) kit (batch number: 20230523), and low-density lipoprotein cholesterol (LDL-C) kit (batch number: 20230525) are all purchased from Nanjing Jiancheng Bioengineering Institute.

[0047] Reverse Transcription Kit (Catalog No.: BL696A, purchased from Biosharp), Testosterone (T) Kit (Catalog No.: MM-0569M1), Luteinizing Hormone (LH) Kit (Catalog No.: MM-44039M1), and Follicle-Stimulating Hormone (FSH) Kit (Catalog No.: MM-45654M1) were all purchased from Jiangsu Enzyme Immunoassay Industry Co., Ltd. (Batch No.: 202408).

[0048] (2) Preparation and grouping of obese oligoasthenospermia model mice:

[0049] Sixty male C57BL / 6J mice weighing 18 - 20 g were randomly divided into a normal group, a model group, a Wuzi Yanzong Pills group (positive group, 1.05 g / kg), and low (1.72 g / kg), medium (34.84 g / kg), and high-dose groups (69.68 g / kg) of Huangjing Yulin Decoction. Except for the normal group, the other groups were fed a high-fat diet for 12 consecutive weeks to prepare an obese oligoasthenospermia model. During the modeling process, drugs were administered by gavage according to the doses of each group. The normal group and the model group were given an equal volume of distilled water. The body weights of the animals in each group were weighed once a week. Thirty minutes after the last drug administration, the mice were anesthetized with sodium barbital (10 mg / kg), blood was collected by eye socket puncture, and then the mice were sacrificed by cervical dislocation. The testes and epididymides were separated for relevant index detection. The animal experiment was reviewed and approved by the Animal Experiment Ethics Committee of Anhui University of Chinese Medicine. SPF-grade C57BL / 6J mice (10 - 20 g) were purchased from Hangzhou Ziyuan Laboratory Animal Technology Co., Ltd., and the animal production license number is SCXK(Zhe)2019 - 0004.

[0050] (3) Body weight measurement, calculation of Lee's index, and organ coefficients of testes, epididymides, etc.

[0051] [[ID=I2]]The body weights of the mice were weighed once every seven days, and the changes in body weights were recorded; at the end of the experiment, the body length (the length of the mouse from the nose to the anus) was measured after anesthesia, and Lee's index was calculated according to the formula [Lee’s index = body weight (g)1 / 3×103 / body length (cm)]; the testes, epididymides, heart, liver, spleen, lungs, and kidneys of the mice were weighed to calculate the organ coefficients. [Organ coefficient = organ weight (g) / body weight (g) × 100].

[0052] (4) Sperm count, sperm motility rate, and sperm vitality determination

[0053] Wash the epididymis, remove the adipose tissue, place it in a beaker containing 3 mL of pre-warmed physiological saline at 37 °C, make 6 - 8 transverse and longitudinal cuts along the tail end of the epididymis with an ophthalmic scissors, disperse it in a 37 °C water bath incubator for 15 min to free the sperm from the tail of the epididymis, filter through a 200-mesh sieve to obtain a sperm suspension, and maintain the temperature at 37 °C. Dip a drop of the sperm suspension and place it in the technical pool in the center of the Marker sperm counting plate, and observe the number of sperm (n) in the 10×10 grid under a 200-fold optical microscope. The number of sperm per milliliter is n×10 6 . Observe 200 sperm for each mouse and determine the sperm motility.

[0054] The grading is as follows: (a): Rapid straight forward movement, straight movement; (b): Slow forward straight movement or sluggish forward movement, slow movement; (c): Non-forward movement or tremors in place, movement in place; (d): Unable to move, non-movement.

[0055] Calculation formula: Sperm motility rate = (a + b + c) / (a + b + c + d)×100%;

[0056] Sperm vitality = (a + b) / (a + b + c + d)×100%.

[0057] (5) Determination of blood lipid levels and sex hormone contents

[0058] After fixation, make the right eyeball of the mouse congested and protruding, cut off the mouse's whiskers to prevent hemolysis when blood flows out, quickly remove the right eyeball of the mouse with an ophthalmic forceps, and let the blood flow into a sterile EP tube. Let it stand at room temperature for 2 h. After stratification, centrifuge at 3000 rpm for 15 min, and take the upper serum to detect the contents of total cholesterol (TC), triacylglycerol (TG), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C). Determine the hormone levels of testosterone (T), luteinizing hormone (LH), and follicle-stimulating hormone (FSH) in the serum according to the ELISA kit instructions.

[0059] (6) Morphological observation of mouse testicular tissue

[0060] Use HE staining to observe the morphology of seminiferous tubules, the area of seminiferous tubules, the status of spermatogenesis, etc. (2) After taking frozen sections of testicular tissue, perform oil red staining to observe lipid deposition in the testis.

[0061] (7) Western blot

[0062] Weigh 20 mg of mouse testicular tissue and place it in a homogenizer. Add 200 μL of RIPA lysis buffer to lyse the myocardial tissue on ice for 30 min. Homogenize until the solution is clear, then transfer it to an EP tube and centrifuge at 12,000 rpm for 15 min at 4 °C to collect the supernatant. Add loading buffer to the collected supernatant according to the ratio and boil it in boiling water for 6 min. Then perform electrophoresis on a 10% SDS-PAGE gel and transfer the protein to an NC membrane. The transferred NC membrane is blocked in 5% skim milk at 4 °C for two hours. Then incubate the NC membrane with different primary antibodies (dilution ratio = 1:1000) overnight at 4 °C. The next day, wash the membrane 3 times in TBST, then incubate it in the secondary antibody (dilution ratio = 1:20000) for two hours. After washing, add ECL luminescent solution and develop the color using a Tianneng 5200 imaging system. Analyze the gray value of the bands using ImageJ.

[0063] The antibodies used in the experiment, GAPDH Rabbit pAb (380626) and CD36 Rabbit pAb (381350), were purchased from Chengdu ZENBIO Biotechnology Co., Ltd. cGAS Antibody (PQA3430), Bcl-2 Antibody (T40056), and Caspase 3 Antibody (TA6311) were all purchased from Abmart Shanghai Co., Ltd. STING Antibody (DF12090) was purchased from Jiangsu Affinity Biological Research Center Co., Ltd., and (Affinity) PPAR-γ (WL01800) was purchased from Shenyang Wanlei Biotechnology Co., Ltd.

[0064] (8) Total RNA extraction from testicular tissue and RNA-Seq analysis

[0065] Three mice were selected from the normal group, model group, and treatment group respectively, and their testicular tissues were excised. Wuhan BGI Tech Co., Ltd. was commissioned to perform RNA extraction and RNA-Seq sequencing. Total RNA was extracted at low temperature using TRIzol lysis buffer. After passing the quality inspection, mRNA was enriched using oligo(dT) magnetic beads. Second-strand cDNA was synthesized using the fragmented mRNA as a template. Subsequently, after denaturing the PCR products into single strands, a circularization reaction system was prepared and the reaction program was set to obtain single-stranded circular products, and the uncircularized linear DNA molecules were digested. Finally, the single-stranded circular DNA molecules were amplified by rolling circle replication to form a DNA nanoball (DNB) containing multiple copies. The obtained DNBs were added into the mesh pores on the chip using high-density DNA nanochip technology, and sequencing was performed through the combinatorial probe-anchor synthesis (cPAS) technology.

[0066] (9) Transcriptome analysis method

[0067] Data filtering:

[0068] The raw data obtained by sequencing was filtered using the filtering software SOAPnuke (v1.6.5) to remove 1) reads containing adapters (adapter contamination); 2) reads with the content of unknown bases N greater than 1%; 3) low-quality reads (reads with the proportion of bases with quality values lower than 15 accounting for more than 40% of the total number of bases in the reads were considered low-quality reads), and clean data was obtained.

[0069] Denovo assembly and quality assessment:

[0070] Trinity (v2.13.2) was used to perform de novo assembly on the clean reads, and the assembly quality was evaluated through BUSCO.

[0071] Reference gene alignment:

[0072] The obtained clean data was aligned to the reference gene sequence using the Bowtie2 (v2.4.5) software, and the RSEM (v1.3.1) software was used to calculate the expression levels of genes and transcripts.

[0073] CDS and SSR prediction:

[0074] The software Transdecoder (v5.5.0) was used to identify candidate coding regions in Unigene, BLAST was used to align to SwissProt, and Hmmscan was used to search for Pfam protein homologous sequences to predict the coding regions. MISA (v1.0) was used to detect Unigene, and then Primer3 (v2.6.1) was used to design primers for the detected SSRs.

[0075] Gene annotation:

[0076] The assembled Unigenes were annotated against seven major functional databases (KEGG, GO, NR, NT, SwissProt, Pfam, and KOG), and transcription factor prediction was performed.

[0077] Time series and WGCNA analysis:

[0078] The software Mfuzz (v2.60.0) was used for time series analysis, and WGCNA (v1.71) was used for gene co-expression network analysis.

[0079] Differentially expressed genes:

[0080] DEseq2 / DEGSeq was used for inter-group differential gene analysis with the conditions of Fold Change ≥ 2 and Adjusted P value ≤ 0.001 (subject to the specific project situation). PoissonDis was used for differential analysis between samples with the conditions of Fold Change ≥ 2 and FDR ≤ 0.001 (subject to the specific project situation). The pheatmap function was used to draw the differential gene clustering heatmap for the differentially expressed gene set. According to the GO and KEGG annotation results and official classification, the differentially expressed genes were functionally classified. The phyper in R software was used for KEGG enrichment analysis, and the Term Finder package was used for GO enrichment analysis. With Q value ≤ 0.05 as the threshold, those meeting this condition were defined as significantly enriched among the candidate genes.

[0081] II. Experimental results

[0082] (1) Effects of Huangjing Yulin Decoction on the transcriptome of obese oligoasthenospermic mice

[0083] Figure 1 It is an image of the effects of Huangjing Yulin Decoction on the transcriptome of obese oligoasthenospermic mice, where Figure 1 a is the statistical chart of the number of differentially expressed genes, Figure 1 b is the VENN diagram of differentially expressed genes, Figure 1 c is the differential gene clustering heatmap, Figure 1 d is the KEGG Pathway enrichment analysis chart of differentially expressed genes.

[0084] Figure 1 A total of 261 genes were shown in a, including 98 up-regulated genes and 163 down-regulated genes. Figure 1 As shown in b, there were 11 intersecting genes in the CTL, M, and HJ groups. Figure 1 Gene expression clustering was shown in c. Figure 1The results showed that the pathways mainly enriched in M / CTL for differential genes included the angiotensin system, necroptosis, apoptosis, etc., indicating that the above pathways were closely related to the pathogenesis of oligoasthenospermia. Enrichment analysis of the differential genes in HJ / M found that they mainly involved the angiotensin system, PPAR signaling pathway, and apoptosis, suggesting that the effect of Huangjing Yulin Decoction on obese oligoasthenospermia might be related to the PPAR signaling pathway and cells.

[0085] (2) Huangjing Yulin Decoction can improve the testicular function of mice with obese oligoasthenospermia

[0086] Figure 2 It is an image of the effect of Huangjing Yulin Decoction on the testicular function of mice with obese oligoasthenospermia. Figure 2 a shows the effect of Huangjing Yulin Decoction on sperm count, motility, and motility rate of mice with obese oligoasthenospermia, Figure 2 b shows the effect of Huangjing Yulin Decoction on TC, TG, LDL, and HDL of mice with obese oligoasthenospermia, Figure 2 c shows the effect of Huangjing Yulin Decoction on T, LH, and FSH of mice with obese oligoasthenospermia, Figure 2 d shows the effect of Huangjing Yulin Decoction on testicular index, epididymal index, and Lee's index (d) of mice with obese oligoasthenospermia. All data are expressed as mean ± SD. *P<0.05, **P<0.01 compared with CON. #P<0.05, ##P<0.01 compared with MODEL.

[0087] To verify the effect of Huangjing Yulin Decoction, tests were conducted in a mouse model of obese oligoasthenospermia. First, the sperm count, sperm motility rate, and vitality of the mice were measured, and compared with the CTL group, all showed significant decreases (P<0.05 or P<0.01). Second, the testicular function of the mice and biochemical indicators such as TC, TG, LDL, HDL, T, FSH, and LH were measured. Compared with the CTL group, the testicular index of the MODEL group increased significantly (P<0.05), and there was no significant difference in the epididymal index. Compared with the CTL group, the Lee's index of the MODEL group increased significantly (P<0.05). In addition, compared with the CTL group, the T level of the MODEL group decreased significantly (P<0.01), while the levels in the Wuzi Yanzong Pills group and the Huangjing Yulin Decoction-H group increased significantly (P<0.05 or P<0.01). Compared with the CTL group, the FSH of the MODEL group increased significantly (P<0.01), and compared with the MODEL group, the levels in the Wuzi Yanzong Pills, Huangjing Yulin Decoction-L, and HZYLD-M groups decreased significantly (P<0.01), and there was no statistical significance for LH. In addition, the TC, TG, and LDL in the serum of the MODEL group mice were significantly higher than those in the CTL group, while the HDL was significantly lower than that in the CTL group (P<0.05 or P<0.01), and after treatment with the Wuzi Yanzong Pills group and each dose group of Huangjing Yulin Decoction, each index recovered significantly (P<0.05 or P<0.01).

[0088] (3) Huangjing Yulin Decoction can improve the testicular structural damage in model mice

[0089] HE staining was used to evaluate the structural damage, and Oil Red O was used to detect lipid deposition. The results are shown in Figure 3 . The HE results showed that the seminiferous tubules of the MODEL group mice were severely atrophied, the number of spermatogonia, interstitial cells, and Sertoli cells decreased significantly, and the number of sperm in the lumen of the testicular tissue decreased. In contrast, it was alleviated after intervention with Wuzi Yanzong Pills and Huangjing Yulin Decoction. The Oil Red O results showed that compared with the CTL group, the lipid deposition in the testis of the MODEL group mice increased significantly; compared with the MODEL group, the lipid deposition in the testis of each dose group of Huangjing Yulin Decoction mice decreased significantly, and the lipid deposition in the testis of the Wuzi Yanzong Pills group mice decreased significantly.

[0090] (4) Western blot

[0091] The protein expression levels of Cgas, Sting, Casepase3, Bcl-2, and Ppar-γ were detected by WB. The results are shown in Figure 4The results showed that compared with the CTL group, the protein expression levels of Cgas, Sting, and Caspase3 in the MODEL group were up-regulated (P<0.05 or P<0.01), and decreased significantly after intervention with Huangjing Yulin Decoction. In addition, the protein expression levels of Bcl-2 and Ppar-γ in the MODEL group were lower than those in the CTL group, while the protein expression levels were significantly increased after intervention with Huangjing Yulin Decoction compared with the model group (P<0.05 or P<0.01).

[0092] Experimental Example 3

[0093] Source of research subjects

[0094] Evaluation of the therapeutic effect of Huangjing Yulin Decoction on obese oligoasthenospermia

[0095] I. Case selection:

[0096] (1) Source of research subjects:

[0097] All cases were from the patients in the Reproductive Andrology Clinic of Chuzhou Hospital of Integrated Traditional Chinese and Western Medicine. The total number of patients with obese oligoasthenospermia diagnosed and confirmed to meet the inclusion criteria was 60. All included cases were randomly assigned to the observation group (Huangjing Yulin Decoction group) and the control group (Wuzi Yanzong Pills group) using the random number table method. Each group had 30 cases, and the cases were strictly selected according to the inclusion criteria.

[0098] (2) Diagnostic criteria:

[0099] Diagnostic criteria for patients with oligoasthenospermia: Refer to the standards of "WHO Laboratory Manual for the Examination and Processing of Human Semen" (5th edition): ① Sperm density < 15×10 6 / mL; ② Sperm motility grade a < 25%, or sperm motility grade a + b < 50%; ③ Survival rate < 60%. All semen sampling and testing were carried out in the treatment room of the Reproductive Andrology Clinic of our hospital. The patients were aged 22 - 45 years, and the disease course was ≥ 1 year.

[0100] Diagnostic criteria for TCM kidney deficiency syndrome: Based on the diagnostic criteria of "TCM Clinical Diagnosis and Treatment Terms - Syndrome Part" and "Guiding Principles for Clinical Research of New Chinese Medicines". Kidney deficiency syndrome: Main symptoms: Male infertility, decreased libido, soreness and weakness of the waist and knees; Sub-symptoms: Dizziness and tinnitus, memory loss, hair loss, inattention, mental fatigue, insomnia and dreaminess, premature ejaculation and spermatorrhea, frequent urination, tongue is redder or light red, tongue coating is thin and white, pulse is deep and thready or thready and rapid. It is necessary to have "infertility" in the main symptoms, and one of the other two in the main symptoms and two in the sub-symptoms to make a diagnosis. Main symptom scoring criteria (each symptom is divided into mild, moderate, and severe degrees, and is recorded as 2, 4, and 6 points respectively, and 0 points if there is no such symptom)

[0101] (3) Inclusion criteria:

[0102] (a) Male patients, aged 22-45 years, with disease duration ≥ 1 year;

[0103] (b) All cases met the diagnostic criteria for oligoasthenospermia and the diagnostic criteria for kidney deficiency syndrome in Traditional Chinese Medicine;

[0104] (c) The subjects or their legal representatives are able to understand the purpose of the study, show sufficient compliance with the study protocol, and sign the informed consent form, with good compliance.

[0105] (4) Exclusion criteria:

[0106] (a) Antisperm antibody positive;

[0107] (b) Those with organic lesions of reproductive organs;

[0108] (c) Those taking other drugs that affect sperm motility;

[0109] (d) Those with severe cardiovascular and cerebrovascular diseases;

[0110] (e) The researcher believes that the subject has other conditions that may affect the clinical trial;

[0111] (f) The spouse of the subject is found to have infertility problems.

[0112] (5) Criteria for exclusion and dropout cases:

[0113] (a) Patients who meet the inclusion criteria but do not actively cooperate with treatment;

[0114] (b) patients who concurrently used or changed other treatment drugs during treatment; case selection did not meet the inclusion criteria and met the exclusion criteria;

[0115] (c) Those with obvious adverse reactions or clinical complications;

[0116] (d) Patients who withdraw on their own;

[0117] (e) Patients with incomplete clinical data.

[0118] 2. Treatment Methods

[0119] (1) Drug preparation

[0120] Huangjing Yulin Decoction: This medicine was provided by the Chinese Medicine Pharmacy Dispensing Group of Chuzhou Hospital of Integrated Traditional Chinese and Western Medicine. It consists of the Chinese medicine compound oral preparation prepared according to Example 1. Decoction is taken orally, 200 mL of the decoction each time, twice daily, one hour after meals.

[0121] Wuzi Yanzong Pills: Produced by Tongrentang Pharmaceutical Factory of Beijing Tongrentang Co., Ltd., approval number: National Medicine Standard Z11020141.

[0122] (2) Dosage:

[0123] Observation group: The patients took Huangjing Yulin Decoction, 1 dose per day, decocted in water for oral administration, and taken in two divided doses, morning and evening.

[0124] Control group: The patients took Wuzi Yanzong Pills, with the administration method: 9 g each time, taken in two divided doses, morning and evening.

[0125] Taking 12 weeks as one treatment course, regularly monitor the ovulation of the female every month, appropriately increase the frequency of sexual intercourse during the ovulation period of the female every month, and appropriately reduce the frequency of sexual intercourse at other times, so as to increase the chance of pregnancy of the female. After 6 weeks and 12 weeks of treatment, semen routine, seminal plasma biochemistry, semen DFI, serum sex hormone content, superoxide dismutase level and other examinations were performed respectively. Abstinence for 3 - 5 days was required before the examination. Subsequently, follow - up was carried out for 3 - 6 months, and the patients visited the doctor once every two weeks. Timely understand and record the clinical efficacy after medication treatment and whether there are any adverse reactions after medication treatment, and require the patients to stop using other drugs related to the treatment of this disease 2 - 4 weeks before the observation and treatment. During the treatment period, the patients were required to have regular work and rest, keep a happy mood, have moderate sexual intercourse, fast from fatty, sweet, thick - flavored foods and spicy and stimulating foods, eat less celery, soy products, and drink less alcohol, coffee, cola, etc., and obtain the cooperation and support of their spouses.

[0126] (3) Observation method

[0127] All patients visited the doctor once every two weeks. After 6 weeks and 12 weeks of treatment, semen routine, seminal plasma biochemistry, semen DFI, serum sex hormone content, superoxide dismutase level and other examinations were re - examined respectively, and their numerical values and related data such as the change scores of TCM clinical symptom volumes were measured and recorded.

[0128] (4) Efficacy judgment criteria:

[0129] The judgment was made according to the "Guiding Principles for Clinical Research of New Chinese Medicines" and relevant efficacy criteria. The results are shown in Table 1. The patients were followed up during the medication period and 3 - 6 months after the treatment, and the pregnancy situation of the patients' spouses was recorded.

[0130] Cured: The clinical symptoms basically disappeared, and the patients' spouses became pregnant;

[0131] Markedly effective: The clinical symptoms were significantly alleviated. Although the patients' spouses did not become pregnant, all the indicators detected in the semen routine examination returned to normal;

[0132] Effective: The clinical symptoms were improved to some extent. The sperm motility detection indicators did not reach normal, but the promotion rate of sperm concentration, grade a sperm or a + b grade sperm was ≥30% compared with that before treatment;

[0133] Ineffective: The clinical symptoms were not improved significantly, and there were no obvious changes in the indicators of sperm concentration and motility in the semen routine examination after treatment.

[0134] Table 1 Therapeutic effect of Huangjing Yulin Decoction on oligoasthenospermia in obese patients

[0135] Group Number of people Cured Markedly effective Effective Ineffective Total effective rate Number of pregnant cases Pregnancy rate Observation group 30 8 12 7 3 90％ 16 53.3％ Control group 30 4 9 8 9 21 6 20％

[0136] As can be seen from the above table, the total effective rate (90.0%) and pregnancy rate (53.3%) of Huangjing Yulin Decoction in the treatment of oligoasthenospermia in obese patients are significantly better than those of Wuzi Yanzong Pills, reflecting the advantages of the present invention in improving sperm quality and increasing the natural pregnancy rate.

[0137] Although the embodiments of the present invention have been shown and described, those of ordinary skill in the art can understand that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

[0138] The above description of the present invention and its embodiments is not restrictive. What is shown in the drawings is only one of the embodiments of the present invention, and the actual application is not limited thereto. Generally speaking, if those of ordinary skill in the art are inspired by it and design similar ways and embodiments without departing from the purpose of the present invention without creative efforts, they shall fall within the protection scope of the present invention.

Claims

1. A traditional Chinese medicine compound oral preparation for treating oligoasthenospermia in men, characterized in that: It is made from the following raw materials in parts by weight: 3 - 35 parts of wine-processed polygonatum sibiricum, 4 - 22 parts of prepared rehmannia root, 3 - 25 parts of cuscuta chinensis, 4 - 23 parts of codonopsis pilosula, 8 - 32 parts of astragalus membranaceus, 3 - 24 parts of poria cocos, 4 - 26 parts of wolfberry fruit, 2 - 22 parts of semen plantaginis (prepared with salt, decocted in a wrapped manner), 3 - 25 parts of dried tangerine peel, 3 - 18 parts of stir-fried atractylodes lancea, 2 - 24 parts of epimedium brevicornu, 3 - 23 parts of stir-fried semen vaccariae.

2. The traditional Chinese medicine compound oral preparation for treating oligospermia and asthenospermia in men according to claim 1, wherein: It is made from the following raw materials in parts by weight: 8 - 25 parts of wine-processed polygonatum sibiricum, 8 - 18 parts of prepared rehmannia root, 8 - 18 parts of cuscuta chinensis, 8 - 18 parts of codonopsis pilosula, 12 - 28 parts of astragalus membranaceus, 8 - 18 parts of poria cocos, 8 - 18 parts of wolfberry fruit, 6 - 18 parts of semen plantaginis (prepared with salt, decocted in a wrapped manner), 8 - 18 parts of dried tangerine peel, 6 - 12 parts of stir-fried atractylodes lancea, 6 - 18 parts of epimedium brevicornu, 8 - 18 parts of stir-fried semen vaccariae.

3. The traditional Chinese medicine compound oral preparation for treating oligospermia and asthenospermia in men according to claim 2, wherein: It is made from the following raw materials in parts by weight: 10 - 22 parts of wine-processed polygonatum sibiricum, 9 - 16 parts of prepared rehmannia root, 9 - 16 parts of cuscuta chinensis, 9 - 16 parts of codonopsis pilosula, 15 - 25 parts of astragalus membranaceus, 9 - 16 parts of poria cocos, 9 - 16 parts of wolfberry fruit, 8 - 16 parts of semen plantaginis (prepared with salt, decocted in a wrapped manner), 9 - 16 parts of dried tangerine peel, 7 - 10 parts of stir-fried atractylodes lancea, 9 - 16 parts of epimedium brevicornu, 9 - 16 parts of stir-fried semen vaccariae.

4. The traditional Chinese medicine compound oral preparation for treating oligospermia and asthenospermia in men according to claim 3, wherein: It is made from the following raw materials in parts by weight: 15 parts of wine-processed polygonatum sibiricum, 10 parts of prepared rehmannia root, 10 parts of cuscuta chinensis, 10 parts of codonopsis pilosula, 20 parts of astragalus membranaceus, 10 parts of poria cocos, 10 parts of wolfberry fruit, 10 parts of semen plantaginis (prepared with salt, decocted in a wrapped manner), 10 parts of dried tangerine peel, 9 parts of stir-fried atractylodes lancea, 10 parts of epimedium brevicornu, 10 parts of stir-fried semen vaccariae.

5. The traditional Chinese medicine compound oral preparation for treating oligospermia and asthenospermia in men according to claim 4, characterized in that: It is made from the following raw materials: 15 g of wine-processed polygonatum sibiricum, 10 g of prepared rehmannia root, 10 g of cuscuta chinensis, 10 g of codonopsis pilosula, 20 g of astragalus membranaceus, 10 g of poria cocos, 10 g of wolfberry fruit, 10 g of semen plantaginis (prepared with salt, decocted in a wrapped manner), 10 g of dried tangerine peel, 9 g of stir-fried atractylodes lancea, 10 g of epimedium brevicornu, 10 g of stir-fried semen vaccariae.

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