Application of liver-soothing kidney-tonifying conceptive decoction in preparation of medicine for treating hypothyroidism type asthenospermia

Abstract

The invention discloses application of liver-soothing and kidney-tonifying conceptive decoction in preparation of a medicine for treating hypothyroidism type asthenospermia. The liver-soothing and kidney-tonifying conceptive decoction is prepared from 12-18 g of tribulus terrestris, 12-18 g of radix curcumae, 12-18 g of fructus lycii, 12-18 g of semen cuscutae, 12-18 g of ligusticum wallichii, 8-12 g of deer-horn glue (molten), 8-12 g of raspberry, 8-12 g of flatstem milkvetch seed and 8-12 g of schisandra chinensis. The liver soothing and kidney tonifying conceptive decoction can repair testis oxidative stress injury, recover thyroid hormone level, regulate endoplasmic reticulum stress PERK / EIF2alpha / ATF4 signal channels and down-regulate pro-apoptotic protein CHOP expression level, so that spermatogenic cell apoptosis is relieved, and a new way is provided for preparation and development of drugs for treating male asthenospermia caused by hypothyroidism.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicines, and particularly relates to an application of Shugan Bushen Yulin Decoction in preparing a medicine for treating hypothyroidism-type asthenospermia. Background Art

[0002] As the second largest endocrine disease, the prevalence of thyroid disease is increasing year by year. As the organ for sperm production and development, the testicle is one of the target organs of thyroid hormone. Studies have found that thyroid hormone imbalance can affect reproductive function, but its mechanism is not yet fully understood. Propylthiouracil (PTU) is a commonly used drug for the treatment of hyperthyroidism. Improper dosage can easily lead to hypothyroidism (abbreviated as hypothyroidism), resulting in abnormal thyroid hormone levels, increased serum thyroid stimulating hormone (TSH) levels, and decreased levels of triiodothyronine (T3) and tetraiodothyronine (T4). Studies have shown that the testicles of rats with hypothyroidism become lighter, the number of germ cells decreases, the seminiferous tubules become thinner and fewer, and sperm parameters deteriorate. Testicular and epididymal damage occurs in rats with hypothyroidism treated with PTU, which is specifically manifested by the degeneration, atrophy and deformation of the seminiferous tubules in the testes, the uneven thickness of the basement membrane, the sparse and disordered arrangement of spermatogonia and spermatocytes at all levels in the tubular lumen, and a significant decrease in number. In severe cases, spermatogenic cells are absent, and the spermatogenic function of rats is reduced; the number of sperm in the epididymal lumen is reduced, the gap area is enlarged, and the basement membrane is thickened. When oxidative stress (OS) occurs in the body, it will affect the development of spermatogenic cells, interstitial cells, and supporting cells and the synthesis of testosterone (T), leading to a decline in male reproductive function. OS has been shown to be the initiator and main contributor of endoplasmic reticulum stress (ERS). In order to restore the homeostasis of the endoplasmic reticulum to maintain cell survival, multiple signaling pathways in the body are activated. When ERS persists, spermatogenic cells apoptosis occurs.

[0003] Shugan Bushen Yulin Decoction is an empirical prescription for treating male infertility. "Clinical Observation on Shugan Bushen Yulin Decoction in Treating Male Infertility Caused by Liver Depression and Kidney Deficiency Asthenospermia"[J] was published in Liaoning Journal of Traditional Chinese Medicine, Vol. 47, No. 10, 2020, pp. 110-112, author Wei Wei. The document shows that Shugan Bushen Yulin Decoction has a good therapeutic effect on male infertility caused by liver depression and kidney deficiency asthenospermia. "Shugan Bushen Yulin Decoction Regulates VDAC2 Gene Methylation to Affect Sperm Mitochondrial Function and Improve Sperm Quality in Rats with Asthenospermia"[A] was published in Chinese Journal of Experimental Traditional Chinese Medicine, Vol. 27, No. 23, December 2021, author Xiao Min. The document shows that Shugan Bushen Yulin Decoction can improve the sperm motility of rats with liver depression and kidney deficiency asthenospermia, which may be through reducing the methylation level of voltage-dependent anion channel 2 (VDAC2) gene, increasing VDAC2 expression, regulating the cAMP / PKA pathway, changing mitochondrial membrane potential, and thus enhancing sperm motility. However, the application of Shugan Bushen Yulin Decoction in the treatment of male asthenospermia caused by hypothyroidism has not yet been found. Summary of the invention

[0004] The purpose of the present invention is to provide the use of Shugan Bushen Yulin Decoction in the preparation of a drug for treating hypothyroidism-type asthenospermia in view of the above-mentioned problems existing in the prior art.

[0005] To achieve the above objectives, the technical solution of the present invention is as follows:

[0006] Application of Shugan Bushen Yulin Decoction in the preparation of drugs for the treatment of hypothyroidism asthenospermia.

[0007] The ingredients of Shugan Bushen Yulin Decoction include: Tribulus terrestris 12-18g, Curcuma 12-18g, Lycium barbarum 12-18g, Cuscuta australis 12-18g, Ligusticum chuanxiong 12-18g, antler glue (melted) 8-12g, Rubus 8-12g, Astragalus membranaceus 8-12g, and Schisandra 8-12g.

[0008] The hypothyroidism-type asthenospermia is testicular spermatogenesis dysfunction and sperm quality decline caused by propylthiouracil-induced hypothyroidism.

[0009] The drug for treating hypothyroidism-type asthenospermia is a preparation for downregulating the expression level of the pro-apoptotic protein CHOP.

[0010] The drug for treating hypothyroidism-type asthenospermia is a preparation for restoring the protein expression level in the PERK / EIF2α / ATF4 signaling pathway.

[0011] The restoration of protein expression levels in the PERK / EIF2α / ATF4 signaling pathway specifically includes: down-regulating the expression levels of p-eIF2a / eIF2a, ATF4, and GRP78.

[0012] The drug for treating hypothyroidism-type asthenospermia is a preparation for restoring thyroid hormone levels.

[0013] The restoration of thyroid hormone levels specifically includes: down-regulating the serum thyroid stimulating hormone (TSH) level, and up-regulating the levels of triiodothyronine (T3), tetraiodothyronine (T4), and testosterone (T).

[0014] The drug for treating hypothyroidism-type asthenospermia is a preparation for repairing oxidative stress.

[0015] The repairing of oxidative stress specifically includes: increasing the activities of SOD and GSH-Px, and down-regulating the level of MDA.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] The present invention explores the application of Shugan Bushen Yulin Decoction in the preparation of a drug for treating hypothyroidism-type asthenospermia. Shugan Bushen Yulin Decoction repairs testicular oxidative damage, regulates the endoplasmic reticulum stress PERK / EIF2α / ATF4 signaling pathway, and downregulates the expression level of the pro-apoptotic protein CHOP, thereby alleviating spermatogenic cell apoptosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The results showed that Shugan Bushen Yulin Decoction improved the gross morphology of the testicles, epididymis, and seminal vesicles in rats.

[0019] Figure 2 The results showed that Shugan Bushen Yulin Decoction improved the pathological changes of rat testicles.

[0020] Figure 3 The results showed that Shugan Bushen Yulin Decoction improved the pathological changes of the rat epididymis.

[0021] Figure 4 is the apoptosis level of testicular spermatogenic cells in rats in the blank group.

[0022] Figure 5 is the apoptosis level of testicular spermatogenic cells in rats in the model group.

[0023] Figure 6 This is the apoptosis level of testicular spermatogenic cells in rats in the low-dose group of Shugan Bushen Yulin Decoction.

[0024] Figure 7 This is the apoptosis level of testicular spermatogenic cells in rats in the medium-dose group of Shugan Bushen Yulin Decoction.

[0025] Figure 8 This is the apoptosis level of testicular spermatogenic cells in rats in the high-dose group of Shugan Bushen Yulin Decoction.

[0026] Fig. 9 is the apoptosis level of testicular spermatogenic cells in rats in L-carnitine group.

[0027] Fig.10 Soothing the Liver and Kidneys and Bushen Yulin Decoction restored the expression levels of GRP78 and CHOP proteins.

[0028] Fig.11 Shugan Bushen Yulin Decoction restored the expression levels of p-PERK, PERK, p-EIF2α, EIF2α, and ATF4 proteins. DETAILED DESCRIPTION

[0029] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0030] Shugan Bushen Yulin Decoction is an empirical prescription, which includes: Tribulus terrestris 12-18g, Curcuma 12-18g, Lycium barbarum 12-18g, Cuscuta chinensis 12-18g, Chuanxiong 12-18g, Deer Horn Glue (melted) 8-12g, Rubus idaeus 8-12g, Astragalus 8-12g, Schisandra 8-12g. Shugan Bushen Yulin Decoction is derived from Wuzi Yanzong Pills, the "first seed prescription in ancient and modern times". On its basis, Plantago asiatica is removed to prevent its penetrating and diuretic properties from damaging kidney qi, which is especially harmful to those with kidney cold and spermatorrhea. Tribulus terrestris, Deer Horn Glue, Chuanxiong, Curcuma zedoaria, and Astragalus asiatica are added to enhance its effects of soothing the liver and relieving depression, and promoting qi and blood circulation. The prescription uses Tribulus terrestris and antler glue as the main ingredients. Tribulus terrestris enters the liver meridian to soothe the liver and relieve depression, and promote the circulation of qi and blood. Deer antler glue enters the liver and kidney meridians to warm and nourish the liver and kidney, and benefit the essence and blood. The two are combined to regulate liver qi, replenish kidney qi, and replenish essence and blood, providing material guarantee for sperm production; Curcuma and Ligusticum chuanxiong promote qi and relieve depression, and help Tribulus terrestris strengthen the function of evacuating qi stagnation, promoting qi and activating blood circulation, and also make the tonification have purgative effects; Lycium barbarum, Cuscuta australis, Rubus idaeus, and Astragalus membranaceus are matched to replenish essence, nourish marrow, and benefit the kidney, providing a material basis for reproductive essence and effectively improving sperm vitality. The four ingredients are collectively the ministers; Schisandra chinensis has the effect of astringency and consolidation to consolidate kidney essence, and can also nourish the kidney and calm the mind, so it is an adjuvant. The combination of various medicines can harmonize the liver and kidney, and both soothe and tonify. Together, they can regulate liver qi, tonify the kidney and benefit essence, harmonize qi and blood, unblock meridians, nourish the tendons, and enable normal sperm production.

[0031] The following research direction is to take ERS as the research direction to explore the intervention effect of Shugan Bushen Yulin Decoction on testicular spermatogenesis dysfunction and decreased sperm quality in hypothyroid rats caused by PTU, in order to improve the testicular spermatogenesis function and sperm quality in hypothyroid rats.

[0032] 1 Materials and Instruments

[0033] 1.1 Animals

[0034] A total of 60 male SPF rats with a body weight of 180-220 g were used. They were purchased from the Hubei Experimental Animal Research Center with a license number of SCXK (E) 2020-0018. They were kept in the Experimental Animal Center of Hubei University of Chinese Medicine in an SPF-grade environment with a constant temperature of (22 ± 2) °C and a relative humidity of 50%-70%. After 7 days of adaptive feeding, subsequent experiments were carried out. The participants in the experiment obtained the Hubei Provincial Professional Technical Examination Certificate for Laboratory Animal Practitioners and were approved by the Animal Experiment Ethics Committee of Hubei University of Chinese Medicine with an ethics approval number of HBUCM00309335.

[0035] 1.2 Drugs and reagents

[0036] Shugan Bushen Yulin Decoction: Tribulus terrestris 15g, Curcuma 15g, Lycium barbarum 15g, Cuscuta chinensis 15g, Chuanxiong 15g, Deer Horn Glue (melted) 10g, Rubus idaeus 10g, Astragalus 10g, Schisandra 10g. All Chinese medicines were purchased from the Chinese medicine pharmacy of Huangjiahu Hospital of Hubei University of Chinese Medicine, and were decocted in accordance with the "Management Standards for Chinese Medicine Decoction Rooms in Medical Institutions". After two decoctions and evaporation, a decoction with a crude drug content of 1.5g / mL was obtained.

[0037] 6-n-propyl-2-thiouracil: purchased from Shanghai Yuanye Biotechnology Co., Ltd., B25353; L-carnitine oral solution: purchased from Shenyang First Pharmaceutical Co., Ltd. of Northeast Pharmaceutical Group, H19990372; superoxide dismutase (SOD) enzyme-linked immunosorbent assay (ELISA) kit, malondialdehyde (MDA) ELISA kit, glutathione peroxidase (GSH-Px) ELISA kit, ECL ultrasensitive luminescent solution, RIPA lysis solution, hematoxylin-eosin (HE) staining kit: all purchased from Beijing Solebow Technology Co., Ltd., BC0175, BC0020 , BC1195, PE0010, R0010, G1421; T3 kit, T4 kit, TSH kit, follicle-stimulating hormone kit (FSH), luteinizing hormone kit (LH), testosterone kit (T), estradiol kit (E2): all purchased from Nanjing Jiancheng Bioengineering Institute, H222-1-2, H223-1-2, H087-1-2, H101-1-2, H206-1-2, H090-1-2, H102-1-2; β-actin monoclonal antibody: purchased from Wuhan ABclonal Company, 9100026001; one-step terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (in situ terminal apoptosis assay) (TUNEL) apoptosis detection kit (FITC green fluorescence), universal tissue fixative, BCA protein quantitative detection kit, SDS-PAGE protein loading buffer: all purchased from Wuhan Saiweier Biotechnology Co., Ltd., G1501, G1101, G2026, G2075; activating transcription factor 4 (ATF4) polyclonal antibody, eukaryotic translation initiation factor 2α kinase (EIF2α) polyclonal antibody, phosphorylated eukaryotic translation initiation factor EIF2α kinase (p-EIF2α) polyclonal antibody, protein kinase R-like endoplasmic reticulum kinase (PERK) polyclonal antibody, phosphorylated protein kinase R-like endoplasmic reticulum kinase (p-PERK) polyclonal antibody, C / EBP homologous protein (CHOP) polyclonal antibody, glucose-regulated protein 78 (GRP78) polyclonal antibody: all purchased from Wuhan Mitaka Biotechnology Co., Ltd., 28657-1-AP, 11170-1-AP, 11170-1-AP, 24390-1-AP, 82534-1-RR, 15204-1-AP, 11587-1-AP.

[0038] 1.3 Instruments

[0039] The KZ-III-F high-speed low-temperature tissue grinder was purchased from Wuhan Sewell Biotechnology Co., Ltd.; the CR21G refrigerated high-speed centrifuge was purchased from Hitachi, Japan; the ES-500 tissue embedding machine was purchased from Shanghai Huanxi Medical Instrument Co., Ltd.; the HS-3345 fully automatic slicer was purchased from Jinhua Huasu Technology Co., Ltd.; the DYCZ-40 electroporator was purchased from Beijing Liuyi Instrument Factory; the E100 microscope and NikonDS-U3 imaging system were both purchased from Nikon Corporation of Japan; the WLJY-9000 color sperm quality detection system was purchased from Beijing Weili Technology Co., Ltd.; and the Multiskan FC microplate reader was purchased from Shanghai Thermo Fisher Scientific Co., Ltd.

[0040] 2 Methods

[0041] 2.1 Animal modeling, grouping and drug administration

[0042] After 7 days of adaptive feeding, 60 SD male rats were randomly divided into a blank group, a model group, a Shugan Bushen Yulin Decoction group (three groups of low, medium and high doses) and a L-carnitine group, with 10 rats in each group. Except for the blank group, the other five groups were intragastrically administered with PTU 15 mg / kg daily, and the dosage was based on the dosage for establishing a rat model of hypothyroidism. After continuous intragastrically administered PTU for 12 days, the “dosage for humans and rats” was converted. The Shugan Bushen Yulin Decoction group was divided into low, medium and high dose groups, and the dosage of Shugan Bushen Yulin Decoction was administered by intragastrically administered with 8.1, 16.2 and 32.4 g / kg, respectively; the L-carnitine group was administered with L-carnitine at a dosage of 0.27 g / kg; 1 ml was administered per 100 g for 4 consecutive weeks, and the model group was intragastrically administered with an equal amount of normal saline.

[0043] 2.2 Body mass and organ index detection

[0044] 24 hours after the last treatment, the rats were weighed and anesthetized with 2% sodium pentobarbital (0.035 g / kg) injected intraperitoneally. Blood was collected and precipitated at room temperature for 2 hours, then centrifuged and the supernatant was placed in a -80°C refrigerator for testing. The testicles, epididymis, and seminal vesicles were removed, the surrounding adipose tissue was removed, and the surface moisture was dried after rinsing with saline. The mass was weighed using a precision balance. The three organ indexes were calculated according to the formula: Testicular index = bilateral testicular mass (mg) / body mass (g) × 100%; epididymal index = bilateral epididymal mass (mg) / body mass (g) × 100%; seminal vesicle index = seminal vesicle mass (mg) / body mass (g) × 100%. The calculation results are shown in Table 2. After weighing, one testicle, epididymis, and seminal vesicle were taken from each group and placed on graph paper for photographing. The photographic results are shown in the following table. Figure 1 shown.

[0045] 2.3 Observation of the pathological morphology of the testis and epididymis and evaluation of the testicular spermatogenesis function of rats using HE method

[0046] One side of the testicle and epididymis tissue was removed from the abdominal cavity of the rat, rinsed with saline and the residual water was absorbed, placed in testis fixative at room temperature for 48 hours, dehydrated with ethanol gradient, then transparentized with xylene, embedded, and finally sliced. After the dewaxing procedure is completed, staining will be carried out immediately, followed by dehydration and permeabilization treatment, and finally sealing. Observe and collect pictures under an optical microscope. The observation results of the testicle and epididymis are as follows: Figure 2 , Figure 3 shown.

[0047] According to the method in "Quantitative Study on Testicular Spermatogenesis in Rat with Varicocele" [J]. Chinese Journal of Pathophysiology, author Yu Jianjun, the spermatogenesis function of rat testis was evaluated. Ten cross-sections of seminiferous tubules were randomly selected under a microscope for each sample. The inner diameter of the seminiferous tubules, the thickness of the tubular membrane, the number of germ cell layers in the tubular wall, and the degree of maturity of the germ cells were measured. Each item was scored from 0 to 5 points, with a full score of 20. The detailed scoring criteria are shown in Table 1. The scoring results are shown in Table 3.

[0048] Table 1 Score table of spermatogenesis function of rat testicular seminiferous tubules

[0049]

[0050] 2.4 Sperm density and motility test

[0051] Preheat phosphate buffered saline (PBS) to 37°C in advance, remove the epididymal head, cut it into pieces and place it in PBS, incubate it in a 37°C water bath for 1 hour, filter it with a 200-mesh sieve to filter out tissue debris, separate sperm, make a sperm suspension for standby use, and then use an automatic sperm analyzer to analyze sperm density and motility. The test results are shown in Table 4.

[0052] 2.5 ELISA method was used to detect the levels of thyroid stimulating hormone (TSH), thyroid hormone (T), triiodothyronine (T3), thyroxine (T4), follicle stimulating hormone (FSH), luteinizing hormone (LH), and estradiol (E2)

[0053] Take out the prepared serum from the -80℃ refrigerator, thaw at room temperature, and follow the ELISA kit steps to test serum TSH, T3, T4, FSH, LH, T, and E2 levels. All procedures were performed on the same day to avoid repeated freezing and thawing of serum. The test results are shown in Table 4.

[0054] 2.6 ELISA was used to detect serum superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and malondialdehyde (MDA) levels

[0055] The prepared serum was taken out from the -80℃ refrigerator, thawed at room temperature, and then the serum SOD, GSH-Px activity and MDA level were determined according to the ELISA kit steps. All procedures were performed on the same day to avoid repeated freezing and thawing of serum. The test results are shown in Table 7.

[0056] 2.7 Detection of apoptosis of testicular spermatogenic cells by TUNEL

[0057] Testicular tissue paraffin sections were taken and operated according to the TUNEL kit instructions. After dewaxing, rinsing, incubation, hematoxylin counterstaining and sealing, testicular tissue cells were observed under a fluorescence microscope and photographed (cell nuclei showing fluorescent green are apoptotic cells). The observation results of the blank group, model group, Shugan Bushen Yulin Decoction low, medium, and high dose groups, and L-carnitine group are shown as follows: Figures 4 to 9 The number of apoptotic cells and the total number of cells were counted, and 6 fields of view were randomly selected from each slice, and the average value was taken as the measured value for comparison, and the apoptosis rate (AI) was calculated using the formula AI = (number of apoptotic cells / total number of cells) × 100%. The statistical results are shown in Table 8.

[0058] 2.8 Western blotting was used to detect the expression levels of testicular activating transcription factor 4 (ATF4), eukaryotic translation initiation factor 2α kinase (EIF2α), phosphorylated eukaryotic translation initiation factor 2α kinase (p-EIF2α), protein kinase R-like endoplasmic reticulum kinase (PERK), phosphorylated protein kinase R-like endoplasmic reticulum kinase (p-PERK), C / EBP homologous protein (CHOP), and glucose-regulated protein 78 (GRP78) pathways

[0059] 50 mg of testicular tissue was taken, and the protein was prepared by lysis and quantitative preparation. After loading, transferring and blocking the extracted protein, the corresponding primary antibody (1:1000) was added and incubated at 4°C overnight; washed again, incubated with secondary antibody (1:3000), and after washing the membrane, it was developed with ECL luminescent liquid, and the band measurement results were photographed with a scanner, and the protein expression level was quantitatively analyzed using Image J software. The electrophoresis diagram of GRP78 and CHOP protein expression is shown in Figure 2. Fig.10 The protein expression levels are shown in Table 9. The electrophoresis diagram of p-PERK, PERK, p-EIF2α, EIF2α, and ATF4 protein expression is shown in Fig.11 The protein expression levels are shown in Table 10.

[0060] 2.9 Statistical analysis

[0061] SPSS 26.0 software was used for statistical analysis. The measured data that conformed to the normal distribution were expressed as mean ± standard deviation. The data were described in percentages, and the differences were statistically significant when P<0.05.

[0062] 3 Results

[0063] 3.1 Effects of Shugan Bushen Yulin Decoction on the gross morphology and organ indexes of rat testis, epididymis, and seminal vesicle

[0064] See also Figure 1 In the figure, A is the blank group, B is the model group, C is the low-dose group of Shugan Bushen Yulin Decoction, D is the medium-dose group of Shugan Bushen Yulin Decoction, E is the high-dose group of Shugan Bushen Yulin Decoction, and F is the L-carnitine group. The background for the photo is a grid paper (the length and width of the grid are both 0.4 cm); Figure 1 It can be seen that compared with the blank group, the volume of the testicles, epididymis and seminal vesicles of the rats in the model group was reduced, and the gross morphology of the testicles, epididymis and seminal vesicles of the rats in the three groups of low, medium and high doses of Shugan Bushen Yulin Decoction was slightly improved.

[0065] See Table 2. Compared with the blank group, the organ indexes of the testis, epididymis and seminal vesicle in the model group decreased (P<0.01), and the low, medium and high doses of Shugan Bushen Yulin Decoction groups increased compared with the model group (P<0.05). Compared with the blank group *P<0.05, **P<0.01; compared with the model group #P<0.05, ##P<0.01.

[0066] Table 2 Effects of Shugan Bushen Yulin Decoction on testis, epididymis and seminal vesicle indexes in rats

[0067]

[0068] 3.2 Effects of Shugan Bushen Yulin Decoction on pathological changes of testis and epididymis and testicular spermatogenesis in rats

[0069] See also Figure 2 , the seminiferous tubules of the rats in the blank group had a compact distance between the tubules, visible interstitial cells, and a regular elliptical tubular morphology. The spermatogenic cells in the lumen were arranged in layers, with spermatogonia visible in the outermost layer, followed by primary spermatocytes, secondary spermatocytes, spermatids, and developing sperm in the inner layer, and mature sperms in the center of the lumen; the seminiferous tubules of the rats in the model group had a larger distance between the tubules, a decrease in interstitial cells, and the spermatogenic cells in the lumen were destroyed and arranged in a disordered manner. In severe cases, spermatogenic cells were missing and no mature sperms were seen; after administration, it can be seen that the distance between the seminiferous tubules of the low, medium, and high dose groups of Shugan Bushen Yulin Decoction was shortened, the arrangement of spermatogenic cells was improved, and the improvement effect was significantly improved with the increase in dose, and even mature sperms appeared in the lumen of the seminiferous tubules in the high dose group.

[0070] See also Figure 3 A large number of mature sperms appeared in the epididymis of the blank group rats, and the sperms were evenly distributed; the sperm distribution in the model group was significantly reduced; low, medium and high doses of Shugan Bushen Yulin Decoction all showed improvement after administration.

[0071] As shown in Table 3, the testicular spermatogenesis function of rats in the blank group was normal; compared with the blank group, the testicular spermatogenesis function of rats in the model group was reduced (P<0.01); compared with the model group, the testicular spermatogenesis function of rats in the low, medium and high dose groups of Shugan Bushen Yulin Decoction was significantly improved, and the difference was statistically significant (P<0.01), and the L-carnitine group also showed an increase (P<0.05).

[0072] Table 3 Effect of Shugan Bushen Yulin Decoction on spermatogenesis in rats

[0073]

[0074]

[0075] 3.3 Effect of Shugan Bushen Yulin Decoction on Sperm Density and Sperm Motility in Rats

[0076] As shown in Table 4, compared with the blank group, the sperm density and sperm motility of rats in the model group were significantly decreased (P<0.01); compared with the model group, the sperm density and sperm motility of the low- and medium-dose Shugan Bushen Yulin Decoction groups were increased (P<0.05), and the sperm density of the high-dose Shugan Bushen Yulin Decoction group and the L-carnitine group was significantly increased (P<0.01).

[0077] Table 4 Effect of Shugan Bushen Yulin Decoction on sperm density and sperm motility in rats

[0078]

[0079] 3.4 Effect of Shugan Bushen Yulin Decoction on Serum TSH, T3, and T4 Levels in Rats

[0080] As shown in Table 5, compared with the blank group, the serum TSH level of rats in the model group increased (P<0.01), the serum T3 level of rats decreased (P<0.01), and the serum T4 level of rats decreased (P<0.05). The serum TSH, T3, and T4 levels of rats in the other four groups were not statistically significant compared with those in the blank group and the model group.

[0081] Table 5 Effects of Shugan Bushen Yulin Decoction on serum TSH, T3 and T4 levels in rats

[0082]

[0083]

[0084] 3.5 Effect of Shugan Bushen Yulin Decoction on Serum FSH, LH, T, and E2 Levels in Rats

[0085] As shown in Table 6, compared with the blank group, the serum FSH, LH, T and E2 levels of the rats in the model group were significantly decreased (P<0.01); compared with the model group, the serum FSH, LH and E2 levels of the rats in the three Shugan Bushen Yulin Decoction groups were increased, the serum E2 level of the rats in the L-carnitine group was increased, the serum T level of the rats in the low- and medium-dose Shugan Bushen Yulin Decoction groups and the L-carnitine group was increased (P<0.05), and the serum T level of the rats in the high-dose Shugan Bushen Yulin Decoction group was significantly increased (P<0.01).

[0086] Table 6 Effects of Shugan Bushen Yulin Decoction on serum FSH, LH, T and E2 levels in rats

[0087]

[0088] 3.6 Effect of Shugan Bushen Yulin Decoction on the activities of serum SOD, GSH-Px and MDA levels in rats

[0089] As shown in Table 7, compared with the blank group, the serum MDA level of rats in the model group was significantly increased, and the activities of SOD and GSH-Px were significantly decreased (P<0.01); compared with the model group, the serum MDA level of rats in the low-dose and medium-dose Shugan Bushen Yulin Decoction groups and L-carnitine group was decreased, the serum GSH-Px activity of rats in the low-dose and medium-dose Shugan Bushen Yulin Decoction groups and L-carnitine group was increased, and the serum SOD activity of rats was increased (P<0.05); the serum MDA level of rats in the high-dose Shugan Bushen Yulin Decoction group was significantly decreased, and the GSH-Px activity was significantly increased (P<0.01).

[0090] Table 7 Effects of Shugan Bushen Yulin Decoction on serum SOD, GSH-Px activities and MDA levels

[0091]

[0092]

[0093] 3.7 Effect of Shugan Bushen Yulin Decoction on the apoptosis level of spermatogenic cells in rats

[0094] See also Figures 4 to 9 It can be seen that most of the testicular cell apoptosis in the model group rats occurred in spermatogonia. In addition, primary and secondary spermatocytes also showed apoptosis, while only a small amount of testicular cells in the blank group rats showed apoptosis.

[0095] As shown in Table 8, compared with the blank group, the apoptosis of testicular cells in the model group was significantly increased (P<0.01). Compared with the model group, the apoptosis rates of testicular cells in the low, medium and high groups of Shugan Bushen Yulin Decoction were all decreased (P<0.05), while there was no statistical significance in the L-carnitine group.

[0096] Table 8 Effect of Shugan Bushen Yulin Decoction on the apoptosis level of testicular cells in rats

[0097] Group dose AI(%) Blank group 5.62±0.79 Model Group <![CDATA[7.18±0.84 ** ]]> Shugan Bushen Yulin Decoction low dose group 8.1g / kg <![CDATA[5.92±0.67 # ]]> Shugan Bushen Yulin Decoction Medium Dose Group 16.2g / kg <![CDATA[5.97±1.04 # ]]> Shugan Bushen Yulin Decoction high dose group 32.4g / kg <![CDATA[5.95±0.88 # ]]> L-Carnitine Group 0.27g / kg 6.12±0.75 F 4.157 P 0.003

[0098] 3.8 Effect of Shugan Bushen Yulin Decoction on the protein expression levels of GRP78 and CHOP in rat testicular tissue

[0099] As shown in Table 9, compared with the blank group, the protein expression levels of GRP78 and CHOP in the testis of the rats in the model group were significantly increased (P<0.01), the protein expression levels of GRP78 and CHOP in the testis of the rats in the low-dose Shugan Bushen Yulin Decoction group were decreased (P<0.05), the protein expression levels of GRP78 and CHOP in the medium- and high-dose Shugan Bushen Yulin Decoction groups were significantly decreased (P<0.05), while no statistical difference was found in the L-carnitine group.

[0100] Table 9 Effect of Shugan Bushen Yulin Decoction on the protein expression levels of GRP78 and CHOP in rat testicular tissue

[0101] Group dose GRP78 CHOP Blank group 0.40±0.02 0.54±0.11 Model Group <![CDATA[0.84±0.13 ** ]]> <![CDATA[1.03±0.20 ** ]]> Shugan Bushen Yulin Decoction low dose group 8.1g / kg <![CDATA[0.51±0.08 # ]]> <![CDATA[0.69±0.03 # ]]> Shugan Bushen Yulin Decoction Medium Dose Group 16.2g / kg <![CDATA[0.44±0.10 ## ]]> <![CDATA[0.54±0.10 ## ]]> Shugan Bushen Yulin Decoction high dose group 32.4g / kg <![CDATA[0.46±0.13 ## ]]> <![CDATA[0.55±0.12 ## ]]> L-Carnitine Group 0.27g / kg 0.58±0.11 0.71±0.09 F 7.773 7.451 P 0.002 0.002

[0102] 3.9 Effect of Shugan Bushen Yulin Decoction on protein expression levels of p-PERK, PERK, p-EIF2α, EIF2α, and ATF4 in rat testicular tissue

[0103] As shown in Table 10, compared with the blank group, the expression levels of p-PERK / PERK, p-eIF2a / eIF2a, and ATF4 proteins in the testicular tissue of rats in the model group were significantly increased (P<0.01); compared with the model group, the expression levels of p-PERK / PERK proteins in the low, medium, and high dose groups of Shugan Bushen Yulin Decoction were all decreased, and the expression levels of p-eIF2a / eIF2a and ATF4 proteins in the low and medium dose groups of Shugan Bushen Yulin Decoction were decreased (P<0.05); the expression levels of p-eIF2a / eIF2a and ATF4 proteins in the high dose group of Shugan Bushen Yulin Decoction were significantly decreased (P<0.01), while no statistical difference was found in the L-carnitine group.

[0104] Table 10 Effect of Shugan Bushen Yulin Decoction on protein expression levels of p-PERK / PERK, p-EIF2α / EIF2α, and ATF4 in rat testicular tissue

[0105]

[0106] 4 Results Analysis

[0107] (1) After PTU intervention, the organ indexes of the testis, epididymis, and seminal vesicle of the model group rats, as well as the sperm density and sperm motility rate, were lower than those of the blank group. Compared with the blank group rats, the number of spermatogenic cells in the testis of the model group rats decreased, and the seminiferous tubules became thinner and fewer. At the same time, due to the correlation between thyroid hormone levels and sperm quality, the reduction of thyroid hormones will also lead to a decrease in the number of proliferating cell nuclear antigen (PCNA) positive cells, inhibiting the proliferation of germ cells and causing damage to the sperm production process.

[0108] Intervention with Shugan Bushen Yulin Decoction can restore the organ indexes of the three and improve sperm quality.

[0109] (2) After PTU intervention, the serum TSH level of the model group rats increased, and the serum T3 and T4 levels decreased, indicating that PTU has induced hypothyroidism in rats. The increase in serum TSH level in rats is due to the loss of negative feedback of the hypothalamus-pituitary-thyroid axis. At the same time, after PTU intervention, the serum FSH, LH, T, and E2 levels of the model group rats decreased. Since spermatogenesis depends on the synergistic effect of FSH, LH, and T, FSH acts on spermatogonia to promote their proliferation and differentiation; T is responsible for the differentiation of spermatogenic cells into sperm and the maturation of sperm in the epididymis, and although LH does not directly act on spermatogenic cells, LH can stimulate interstitial cells to secrete T. The three coordinate to promote spermatogenesis and maturation; E2 affects spermatogenesis, and the thyroid gland also affects the level of E2; the level of free T in rat serum decreased, which may be due to OS caused by hypothyroidism, which in turn causes damage to interstitial cells.

[0110] After intervention with Shugan Bushen Yulin Decoction, the thyroid hormone level was reversed, thus improving spermatogenesis. This may be because Cuscuta flavonoids can increase the levels of T, E2, and LH in rat serum, Lycium barbarum polysaccharides can increase the levels of T, FSH, and LH in mouse serum, and both Astragalus complanatus total flavonoids and Schisandra chinensis polysaccharides can increase the serum T level in rats with spermatogenesis dysfunction.

[0111] (3) After PTU intervention, the MDA level of the model group rats increased, and the activities of antioxidant enzymes and SOD decreased, indicating that OS was activated in the rats. Under OS, the hypothalamus-pituitary-thyroid axis was affected, resulting in abnormal LH and FSH levels, reduced expression of androgen binding protein in supporting cells, affecting T synthesis, and then affecting the level of reproductive hormones in the rats.

[0112] After intervention with Shugan Bushen Yulin Decoction, the MDA level of rats decreased, and the activity of GSH-Px and SOD increased.

[0113] (4) After PTU intervention, ERS occurred in the model group rats. When ERS existed for a long time, the number of unfolded or misfolded proteins in the cells increased. The three transmembrane proteins p-PERK, IRE1α and ATF6 would bind to the unfolded proteins. GRP78 would dissociate from p-PERK, IRE1α and ATF during ERS. After PERK dissociated from GRP78, it activated the downstream EIF2α protein in the form of phosphorylation, which led to a decrease in protein synthesis and prevented the newly synthesized proteins from entering the endoplasmic reticulum. After EIF2α was phosphorylated, it preferentially activated ATF4, which then started the expression of the downstream pro-apoptotic protein CHOP, thereby causing spermatogenic cell apoptosis. The above results showed that PTU activated the ERS-induced apoptotic signaling pathway, and its main pathway was to activate the downstream pro-apoptotic protein CHOP by activating the PERK / EIF2α / ATF4 signaling pathway, thereby inducing apoptosis of rat testicular spermatogenic cells.

[0114] After intervention with Shugan Bushen Yulin Decoction, ERS levels were downregulated and spermatogenic cell apoptosis was improved, which indicates that Shugan Bushen Yulin Decoction can regulate ERS through the PERK / EIF2α / ATF4 signaling pathway and improve ERS-induced spermatogenic cell apoptosis in rat testis.

[0115] In summary, Shugan Bushen Yulin Decoction can improve testicular spermatogenesis dysfunction and sperm quality in rats with hypothyroidism induced by PTU. Its intrinsic mechanism is to repair testicular oxidative damage and regulate the endoplasmic reticulum stress PERK / EIF2α / ATF4 signaling pathway, thereby alleviating spermatogenic cell apoptosis.

[0116] The above description is only a preferred embodiment of the present invention, and does not limit the implementation mode and protection scope of the present invention. For those skilled in the art, it should be aware that all solutions obtained by equivalent substitutions and obvious changes made using the description and illustrations of the present invention should be included in the protection scope of the present invention.

Claims

1. Application of Shugan Bushen Yulin Decoction in the preparation of drugs for the treatment of hypothyroidism-type asthenospermia.

2. The use according to claim 1, characterized in that: The ingredients of Shugan Bushen Yulin Decoction include: Tribulus terrestris 12-18g, Curcuma 12-18g, Lycium barbarum 12-18g, Cuscuta australis 12-18g, Ligusticum chuanxiong 12-18g, antler glue (melted) 8-12g, Rubus 8-12g, Astragalus membranaceus 8-12g, and Schisandra 8-12g.

3. The use according to claim 1, characterized in that: The hypothyroidism-type asthenospermia is testicular spermatogenesis dysfunction and sperm quality decline caused by propylthiouracil-induced hypothyroidism.

4. The use according to claim 3, characterized in that: The drug for treating hypothyroidism-type asthenospermia is a preparation for downregulating the expression level of the pro-apoptotic protein CHOP.

5. The use according to claim 4, characterized in that: The drug for treating hypothyroidism-type asthenospermia is a preparation for restoring the protein expression level in the PERK / EIF2α / ATF4 signaling pathway.

6. The use according to claim 5, characterized in that: The restoration of protein expression levels in the PERK / EIF2α / ATF4 signaling pathway specifically includes: down-regulating the expression levels of p-eIF2a / eIF2a, ATF4, and GRP78.

7. The use according to claim 3, characterized in that: The drug for treating hypothyroidism-type asthenospermia is a preparation for restoring thyroid hormone levels.

8. The use according to claim 7, characterized in that: The restoration of thyroid hormone levels specifically includes: down-regulating the serum thyroid stimulating hormone (TSH) level, and up-regulating the levels of triiodothyronine (T3), tetraiodothyronine (T4), and testosterone (T).

9. The use according to claim 3, characterized in that: The drug for treating hypothyroidism-type asthenospermia is a preparation for repairing oxidative stress.

10. The use according to claim 9, characterized in that: The repairing of oxidative stress specifically includes: increasing the activities of SOD and GSH-Px, and down-regulating the level of MDA.

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