Application of semen cuscutae-fructus lycii in preparation of medicine for regulating SIRT1 / Nrf2 signal channel to improve oligoasthenospermia

Abstract

The invention discloses application of semen cuscutae-fructus lycii in preparation of a medicine for regulating and controlling an SIRT1 / Nrf2 signal channel to improve oligoasthenospermia, and belongs to the technical field of biology. According to the application of the semen cuscutae-fructus lycii in preparing the medicine for regulating and controlling the SIRT1 / Nrf2 signal channel to improve the oligoasthenospermia, disclosed by the invention, the semen cuscutae-fructus lycii regulates and controls the SIRT1 / Nrf2 expression of testicular tissues, enhances the anti-oxidative stress capability, up-regulates the BTB related protein expression level and repairs BTB damage, so that the spermatogenic function of OAS rats is improved, and the effect of improving the oligoasthenospermia is achieved. And the total flavonoids can be a material basis for playing a drug treatment effect. A powerful experimental support is provided for research on a mechanism of treating OAS by exerting the anti-oxidative stress effect of the semen cuscutae-fructus lycii, and a theoretical basis is provided for research on Ningxia genuine medicinal materials.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and more specifically to the use of Cuscuta seeds and Lycium barbarum in the preparation of a drug for regulating SIRT1 / Nrf2 signaling pathway to improve oligoasthenospermia. Background Art

[0002] Oligospermia (OAS) is the most common type of male infertility, mainly characterized by sperm concentration <15×10 6 mL -1 or total sperm count < 39 × 10 6 mL -1 and total sperm motility (PR+NP) <40% or forward motility (PR) <32%.

[0003] At present, there is no specific treatment for OAS, and the pathogenesis is still unclear. Related studies have shown that oxidative stress can cause testicular tissue damage, leading to a decrease in sperm count and motility, so anti-oxidative stress treatment has gradually attracted attention from the academic community. The blood-testis barrier (BTB) is located between adjacent supporting cells in the seminiferous tubules. It is composed of tight junctions, gap junctions, and adhesion junctions, providing a stable microenvironment for the development and maturation of germ cells. When the body is stimulated by external stimuli and induces testicular oxidative stress, it can affect cell junction proteins, thereby destroying the integrity of the BTB, ultimately leading to a decrease in sperm quality and causing male infertility. SIRT1 / Nrf2 is an important pathway for regulating oxidative stress response. Activating the SIRT1 / Nrf2 pathway can reduce blood-testis barrier damage caused by oxidative stress. When the SIRT1 / Nrf2 pathway is inhibited, as the expression levels of structural proteins such as ZO-1 and occludin decrease, the BTB function is impaired and spermatogenesis is affected. Therefore, inhibiting oxidative stress damage and repairing the integrity of the BTB are treatment approaches that can be tried to improve the spermatogenesis of OAS rats.

[0004] The etiology of OAS is complex, and the pathological formation mechanism is still unclear. Epidemiological studies suggest that it is one of the main causes of male infertility, and the incidence rate is increasing year by year. At present, the indications and efficacy of surgical treatment of OAS are relatively limited. Although drugs such as L-carnitine, coenzyme Q10, and vitamin E can improve semen quality to a certain extent, they lack high-level evidence-based medicine. Traditional Chinese medicine has a long history of treating OAS, with the advantages of definite clinical efficacy and few side effects. The disease can be classified into the categories of "childlessness", "low sperm count", and "thin sperm count" in traditional Chinese medicine, and "yang deficiency and sperm deficiency" is its main syndrome type, and "warming and tonifying kidney essence" is its key treatment principle.

[0005] Therefore, providing the use of Cuscuta australis-Wolfberry in the preparation of drugs for regulating SIRT1 / Nrf2 signaling pathway to improve oligoasthenospermia is an urgent problem to be solved by those skilled in the art. Summary of the invention

[0006] In view of this, the present invention provides the use of Cuscuta chinensis-Wolfberry in the preparation of drugs for regulating SIRT1 / Nrf2 signaling pathway to improve oligoasthenozoospermia, studies the expression of SIRT1 / Nrf2 pathway in the testicular tissue of OAS rats, and evaluates the mechanism and material basis of "Cuscuta chinensis-Wolfberry" in alleviating oxidative stress response of testicular tissue, restoring blood-testis barrier dysfunction and improving spermatogenesis, so as to provide theoretical research basis and ideas for the treatment of OAS.

[0007] In order to achieve the above object, the present invention adopts the following technical solution:

[0008] Application of Cuscuta australis and Lycium barbarum in the preparation of drugs for regulating SIRT1 / Nrf2 signaling pathway to improve oligoasthenospermia.

[0009] Furthermore, the application of Cuscuta seeds and Lycium barbarum in the preparation of drugs for inhibiting blood-testis barrier damage after oxidative stress.

[0010] Furthermore, Cuscuta australis-Lycium barbarum is used in the preparation of drugs that regulate the SIRT1 / Nrf2 signaling pathway, inhibit blood-testis barrier damage after oxidative stress, and improve oligoasthenospermia.

[0011] Furthermore, the total flavonoids of Cuscuta australis and Lycium barbarum are used in the preparation of drugs that regulate the SIRT1 / Nrf2 signaling pathway to improve oligoasthenospermia.

[0012] Furthermore, the total flavonoids of Cuscuta australis and Lycium barbarum are used in the preparation of drugs for inhibiting blood-testis barrier damage after oxidative stress.

[0013] Furthermore, the total flavonoids of Cuscuta australis and Lycium barbarum are used in the preparation of drugs that regulate the SIRT1 / Nrf2 signaling pathway, inhibit blood-testis barrier damage after oxidative stress, and improve oligoasthenospermia.

[0014] At present, there are various ways to model OAS animal models, each with its own advantages and disadvantages, and there are certain limitations. According to the clinical characteristics of the disease, an animal model combining "disease and syndrome" can be established, which can provide animal experimental support for drug efficacy evaluation and new drug development. The present invention innovatively uses a single-factor stimulation method combined with hydrocortisone solution gavage to construct an OAS rat "Yang deficiency and sperm deficiency syndrome" model. Based on the theory of "room fatigue and kidney injury", the single-factor stimulation method is used to make rats ejaculate excessively, so as to achieve the purpose of kidney essence consumption; the hydrocortisone modeling method is simple to operate, has a short modeling cycle, is easy to repeat, and can simulate the symptoms of kidney yin deficiency and kidney yang deficiency. The two methods are combined to jointly construct a stable "Yang deficiency and sperm deficiency syndrome" model. During the model evaluation period, it was found that the sperm count, linearity, swinging, and forwardness of the model rats were gradually lower than those of the blank group as the modeling time was delayed. At 8 weeks after modeling, the seminiferous tubules of the testicular tissue of the model rats were loosely arranged and the morphology was obviously destroyed. Only a very small number of sperm were seen in the lumen of the seminiferous tubules, and the TMS score decreased. After 4 weeks of drug intervention, the sperm count of rats in the Cuscuta-Wolfberry Granules group, Cuscuta-Wolfberry Total Flavonoids group, and L-Carnitine group increased significantly, and the sperm motility of rats in the Cuscuta-Wolfberry Granules group and L-Carnitine group improved. The seminiferous tubule structure of the testicular tissue in these three groups was repaired, the arrangement was regular, the number of spermatogenic cells increased, the number of sperm in the lumen increased, and the TMS score increased. It shows that Cuscuta-Wolfberry can improve the spermatogenic function and sperm quality of OAS rats. Total flavonoids is a quality marker of Cuscuta, and is one of the main material bases for L-Carnitine to exert its pharmacodynamic effect. The present invention also suggests that Cuscuta-Wolfberry Total Flavonoids can repair testicular tissue pathological damage and improve spermatogenic ability, and no relevant research has been reported. The above results suggest that the Cuscuta chinensis-Lycium barbarum drug pair has a good therapeutic effect on OAS rats, can improve the pathological damage of testicular tissue in OAS rats, promote spermatogenesis, and improve sperm quality. At the same time, total flavonoids may be the potential material basis for its effect.

[0015] Oxidative stress can damage connexins, thereby destroying the blood-testis barrier (BTB), leading to the disturbance of the homeostasis of sperm production, causing excessive DNA damage in the sperm formation process, resulting in the total amount of sperm and sperm quality being affected, and finally causing OAS. At present, the advantages of antioxidants in improving the semen quality, sperm motility and quantity of OAS patients are gradually becoming apparent, and have good research prospects. The SIRT1 / Nrf2 pathway is closely related to the body's antioxidant stress level. SIRT1 is an NAD-dependent deacetylase that plays a key role in maintaining mitochondrial function and reducing oxidative damage. It is also the most famous regulator in the Nrf2 signaling pathway. Overexpression of SIRT1 can upregulate Nrf2 expression. After being activated under oxidative stress, Nrf2 enters the cell nucleus and can bind to the antioxidant response element (ARE) to initiate the transcription of downstream gene expression, ultimately playing an antioxidant stress role. Activating the Nrf2 pathway, inhibiting ROS accumulation and cell apoptosis, can upregulate the expression of connexin43 (Cx43) and ZO-1 to prevent the increase of BTB permeability, thereby repairing the testicular microstructure and spermatogenesis disorder. The BTB is located near the base of the seminiferous tubules and is composed of tight junctions, ectoplasmic specialization, desmosomes and gap junctions between supporting cells. It is a barrier necessary for spermatogenesis. Its main function is to isolate cells located in the glandular cavity from the circulation and lymphatic system, and provide an immune privileged microenvironment for the completion of meiosis. Occludin and ZO-1 are tight junction proteins, and adhesion junctions such as β-catenin are located between supporting cells and germ cells. CX-43 is the most abundantly expressed among gap junction proteins. When supporting cells undergo ferroptosis, oxidative stress will lead to a decrease in cell junction proteins such as ZO-1 and CX-43, destroying the blood-testis barrier function and leading to a decrease in spermatogenesis. It can be seen that upregulating SIRT1 / Nrf2 expression can not only inhibit oxidative stress response to damage BTB, but also upregulate the level of cell junction proteins to repair BTB and thus improve the spermatogenesis function of OAS rats.

[0016] During the experiment, it was found that the levels of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in the OAS model group rats decreased, and the level of malondialdehyde (MDA) increased; at the same time, the mRNA transcription and protein translation levels of SIRT1 and Nrf2 decreased; transmission electron microscopy results showed that the tight junctions of the testicular supporting cells of the model group rats were loose, some were broken, the mitochondria were swollen, the mitochondrial cristae were broken, and even vacuoles appeared, indicating that oxidative stress occurred in the testicular tissue of OAS rats and the BTB structure was damaged. After 4 weeks of intervention treatment with Cuscuta-Lycium granules and Cuscuta-Lycium total flavonoids, the levels of GSH-PX and SOD in the testicular tissue of OAS rats increased, and the level of MDA decreased after treatment with Cuscuta-Lycium granules, while the levels of MDA in the Cuscuta-Lycium total flavonoids group and L-carnitine group were higher than those in the model group, indicating that Cuscuta-Lycium can enhance the antioxidant capacity of OAS rats. The antioxidant effects of Cuscuta-Lycium total flavonoids and L-carnitine may be mainly reflected in increasing the activity of antioxidant enzymes GSH-PX and SOD, scavenging free radicals in the body and directly reducing oxidative damage, but are weak in reducing the accumulation of lipid peroxide end product MDA and indirectly enhancing resistance to free radical attack. At the same time, after intervention with Cuscuta-Lycium barbarum granules and total flavonoids, the expression levels of SIRT1 and Nrf2 proteins and mRNA were significantly increased, supporting cell tight junctions and mitochondrial microstructures were repaired to varying degrees, and the expression levels of Occludin, ZO-1, CX-43, and β-catenin proteins and mRNA in testicular tissue increased significantly, indicating that Cuscuta-Lycium barbarum and its total flavonoids can play a role in repairing BTB damage, fully reflecting the advantages of multi-component and multi-target effects of traditional Chinese medicine. In addition, the present invention also found that L-carnitine down-regulated the expression level of SIRT1 protein in the testicular tissue of OAS rats; studies have shown that L-carnitine oral solution repairs oxidative stress damage to testicular tissue by increasing the expression of Nrf2, which is consistent with the increase in the expression level of Nrf2 protein in rat testicular tissue after L-carnitine intervention in this study.

[0017] In summary, the Cuscuta-Wolfberry drug pair regulates the expression of SIRT1 / Nrf2 in testicular tissue, enhances the ability to resist oxidative stress, upregulates the expression level of BTB-related proteins, repairs BTB damage, and thus improves the spermatogenesis function of OAS rats, and total flavonoids may be the material basis for its drug treatment. The present invention provides strong experimental support for the study of the mechanism of Cuscuta-Wolfberry in treating OAS by resisting oxidative stress, and provides a theoretical basis for the study of Ningxia authentic medicinal materials. However, whether the pharmacological effect of Cuscuta-Wolfberry involves other signal pathways that regulate oxidative stress needs further study.

[0018] Through the above technical scheme, it can be known that compared with the prior art, the present invention discloses the use of Cuscuta-Lycium barbarum in the preparation of drugs for regulating SIRT1 / Nrf2 signaling pathway to improve oligoasthenozoospermia, and studies the effect and mechanism of "Cuscuta-Lycium barbarum" drugs on regulating silent information regulator (SIRT1) / nuclear factor E2-related factor 2 (Nrf2) to inhibit blood-testis barrier dysfunction caused by oxidative stress and improve the spermatogenesis function of oligoasthenozoospermia (OAS) rats. 35 male SD rats were randomly divided into a normal group (7 rats) and a modeling group (28 rats). The OAS rat model was established by gavage of hydrocortisone aqueous solution combined with single-factor electrical stimulation. The rats were randomly divided into a model group, a Cuscuta-Lycium barbarum granule group (3.2 g·kg -1 ), Cuscuta seeds-Lycium barbarum total flavonoids group (0.1 g·kg -1 +0.24g kg -1 ) and L-carnitine group (0.38 g·kg -1), and the drug was administered for 4 weeks. The sperm quality of rats was detected by automatic sperm analyzer; the levels of superoxide dismutase (SOD), malondialdehyde (MAD), and glutathione peroxidase (GSH-Px) in rat testis tissue were detected by enzyme-linked immunosorbent assay (ELISA); the pathological changes of testis tissue and spermatogenic function score of rats were observed by hematoxylin-eosin (HE) staining; the ultrastructural changes of testicular supporting cells of rats were observed by transmission electron microscopy (TEM); Western blot and real-time fluorescence quantitative polymerase chain reaction (Real-time PCR) were used to detect the protein expressions and mRNA transcription levels of silent information regulator 1 (SIRT1), nuclear factor E2-related factor 2 (Nrf2), occludin, zonula occludens 1 (ZO-1), connexin43 (CX43), and β-catenin in rats in each group. Results: Compared with the normal group, the total sperm count and motility in the model group were decreased (P<0.01), the testicular tissue and blood-testis barrier structure were significantly damaged, the spermatogenesis score was decreased (P<0.01), the GSH-PX level and SOD level in the testicular tissue were decreased (P<0.05), the MDA level was increased (P>0.05), the expression levels of SIRT1, Nrf2, ZO-1, CX43, β-catenin and occludin proteins in the testicular tissue were decreased to varying degrees (P<0.05, P<0.01), and the mRNA transcription levels of SIRT1, Nrf2, ZO-1, CX43 and β-catenin were decreased (P<0.05, P<0.01). After drug treatment, the testicular tissue structure damage in the Cuscuta-Lycium barbarum granules group, Cuscuta-Lycium barbarum total flavonoids group, and L-carnitine group was improved, and the spermatogenesis function score was increased (P<0.01). Compared with the model group, the levels of GSH-PX and SOD in the treatment groups were increased (P<0.05, P<0.01), and the level of MDA in the Cuscuta-Lycium barbarum granules group was decreased (P>0.05). The protein and mRNA expressions of SIRT1, Nrf2, ZO-1, CX43, β-catenin, and occludin in the testicular tissue of the Cuscuta-Lycium barbarum granules group and Cuscuta-Lycium barbarum total flavonoids group were increased (P<0.05, P<0.01). The Cuscuta seeds-Lycium barbarum drug pair can improve the spermatogenesis function of oligoasthenozoospermia rats. Its mechanism may be related to regulating the SIRT1 / Nrf2 pathway to inhibit oxidative stress and alleviate blood-testis barrier damage. Total flavonoids may be the material basis for the Cuscuta seeds-Lycium barbarum drug pair to exert this therapeutic effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0020] Figure 1 The attached figure shows the effect of the Cuscuta chinensis-Lycium barbarum of the present invention on the pathological morphology of rat testicular tissue (HE, x5, x10); x5: scale bar 300 μm; x10: scale bar 200 μm;

[0021] Among them, A: normal group; B: model group; C: L-carnitine group; D: Cuscuta-wolfberry granules group; E: Cuscuta-wolfberry total flavonoids group;

[0022] Figure 2 The accompanying drawing shows the effect of the dodder seed-wolfberry fruit of the present invention on the ultrastructure of rat testicular supporting cells (x7000);

[0023] Among them, A: normal group; B: model group; D: Cuscuta-Lycium barbarum granules group; E: Cuscuta-Lycium barbarum total flavonoids group;

[0024] Figure 3 The accompanying drawing shows the effect of the Cuscuta seeds-Wolfberry fruit of the present invention on the expression levels of SIRT1 and Nrf2 in rat testicular tissue;

[0025] Among them, A: normal group; B: model group; C: L-carnitine group; D: Cuscuta-wolfberry granules group; E: Cuscuta-wolfberry total flavonoids group;

[0026] Figure 4 The accompanying drawing shows the effect of the Cuscuta seeds-Wolfberry fruit of the present invention on the level of blood-testis barrier-related proteins in rat testicular tissue;

[0027] Among them, A: normal group; B: model group; C: L-carnitine group; D: Cuscuta seeds-wolfberry granules group; E: Cuscuta seeds-wolfberry total flavonoids group. DETAILED DESCRIPTION

[0028] 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.

[0029] animal:

[0030] Thirty-five SPF 6-week-old male rats, weighing (200±20) g, were provided by the Experimental Animal Center of Ningxia Medical University, with animal license number SYXK(Ning)2020-0001. The animals were kept at a temperature of 25-26°C, a humidity of 40%-50%, 12h light-dark alternation, fed with ordinary feed, and had free access to drinking water. The bedding was changed every other day, and the cage was kept clean and dry. This experiment was approved by the Animal Experiment Ethics Committee of Ningxia Medical University, with the ethics number (IACUC-NYLAC-2024-042).

[0031] drug:

[0032] Hydrocortisone acetate tablets (Shanghai SPH Xinyi Pharmaceuticals Co., Ltd., approved by the National Medicine Standard H31021270); L-carnitine oral solution (Northeast Pharmaceutical Group Shenyang No. 1 Pharmaceutical Co., Ltd., approved by the National Medicine Standard H19990372); total flavonoids from Cuscuta seeds (Nanjing Xinhou Biotechnology Co., Ltd., content 80%, batch number XH20240410); total flavonoids from Lycium barbarum (Nanjing Xinhou Biotechnology Co., Ltd., content 80%, batch number XH20240405); Cuscuta seeds granules and Lycium barbarum decoction-free granules were purchased from Ningxia Dayi Traditional Chinese Medicine Hospital, each 1g of Cuscuta seeds granules is equivalent to 5g of Chinese medicine slices, and each 1g of Lycium barbarum granules is equivalent to 0.9g of Chinese medicine slices; Cuscuta seeds granules and Lycium barbarum decoction-free granules were mixed in a ratio of 1:1, dissolved in distilled water, and stored at 4°C for later use.

[0033] Reagents:

[0034] Malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px) ELISA enzyme-linked immunosorbent assay kits (Jiangsu Jingmei Biotechnology Co., Ltd., catalog numbers JM-10323R2, JM-01793R2, and JM-02173R2, respectively). Rabbit primary antibody SIRT1 (Abimat Pharmaceutical Technology (Shanghai) Co., Ltd., catalog number MB0189); rabbit primary antibodies NRF2, Occludin, ZO-1, Connexin 43, mouse primary antibody Beta Catenin, internal reference antibody GAPDH (Wuhan Sanying Biotechnology Co., Ltd., catalog numbers 80593-1-RR, 27260-1-AP, 21773-1-AP, 26980-1-AP, 66379-1-Ig, 60004-1-Ig, respectively); secondary antibody Goat Anti-MOUSE IgG (H&L) (Suzhou Youyi Landi Biotechnology Co., Ltd., catalog number Y6108L), Goat Anti-Rabbit IgG-HRP Antibody (Shanghai Aibixin Biotechnology Co., Ltd., catalog number abs20040ss); internal reference antibody β-Actin (Nanjing Baaode Biotechnology Co., Ltd., catalog number BS6007M). SPARKeasy tissue / cell RNA rapid extraction kit (Shandong Sikejie Biotechnology Co., Ltd., Catalog No. AC0202-A); PrimeScript RT reagent Kit with gDNA Eraser (Perfect Real Time) (Baoriyi Biotechnology Co., Ltd., Catalog No. RR047A); SYBR Green qPCR premix (Wuhan Saiweier Biotechnology Co., Ltd., Catalog No. G3326-01).

[0035] instrument:

[0036] Cryo-grinding machine (JXFSTPRP-CL, Shanghai Jingxin); fluorescence quantitative PCR instrument (Stepone Plus, ABI, USA); protein electrophoresis system (Mini Protean Tetra, Bio-Rad, USA); transfer system (Mini Trans-Blot cel, BIO-RAD, USA); slice scanner (Aperio LV1, Germany / Leica); tissue paraffin embedding machine (ArcadiaH+C, Germany / Leica); slide spreader and baking machine (HI1220, Germany / Leica); microplate reader (Multiskan Go, Thermo Fisher Scientific).

[0037] Statistical analysis

[0038] SPSS 26.0 was used for statistical analysis. The experimental data were quantitative data and were expressed as mean ± standard deviation. The data conformed to normal distribution. One-way analysis of variance was used for comparison among multiple groups. If the variances were equal, the LSD method was used for pairwise comparisons among groups; if the variances were unequal, the Games-Howell method was used for comparisons. The data did not conform to normal distribution, and multiple group rank sum tests were performed, and the median (interquartile range) was used for expression. (P < 0.05 was statistically significant, and P < 0.01 was statistically significant).

[0039] Example 1

[0040] 1) Animal model preparation

[0041] Thirty-five rats were randomly divided into a normal group, a model group, a L-carnitine group, a dodder seed-wolfberry granule group, and a dodder seed-wolfberry total flavonoids group, with 7 rats in each group. After one week of adaptive feeding, the normal group was allowed to eat and drink freely, and 0.04 ml·g -1 ·d -1 Distilled water was administered orally; the other groups were given 50 mg kg / day on the basis of free diet and drinking water. -1 Hydrocortisone aqueous solution was gavaged, and single-factor electrical stimulation was given after an interval of four hours until ejaculation (frequency 50Hz, starting voltage 1V, after 30s of stimulation, the voltage was increased by 1V increments depending on the situation, and the maximum should not exceed 5V, and the operation was repeated until semen was discharged), for 4 consecutive weeks to establish an oligoasthenozoospermia model. Two rats in the normal group and the modeling group were randomly selected from one side of the epididymis for sperm quality analysis every week, and three rats were selected in each group for sperm quality analysis in the fourth week. The decline in sperm quality indicated that the modeling was successful.

[0042] Sperm quality analysis was performed two weeks after modeling, and it was found that the total number of sperm and total number of active sperm in the model group gradually decreased with the extension of modeling time. At the fourth week of modeling, 3 rats were selected from each group for evaluation. The total number of sperm and total number of active sperm in the model group decreased significantly, and the difference between the two groups was statistically significant (P < 0.01), indicating that the modeling was successful. The results are shown in Table 1.

[0043] Table 1 Comparison of sperm quality between two groups of rats

[0044]

[0045] 2) After modeling, drug intervention

[0046] According to the dosage conversion factor between rats and humans in Pharmacological Experimental Methodology, which is 6.3, the oral dose of Cuscuta-Lycium barbarum granules was 3.2 g·kg -1 The oral dose of L-carnitine group was 0.38 g·kg -1 , the Cuscuta Semen-Lycium Barbarum Total Flavonoids group was given (0.1+0.24) g·kg -1The normal group and the model group were given the same volume of distilled water by gavage, and the intervention was carried out daily for a total of 4 weeks. 12 hours after the last administration, testicular tissue was obtained for subsequent detection.

[0047] 3) Automatic analyzer to detect sperm quality

[0048] The rat epididymis was placed in a culture dish, rinsed with saline, and placed in a 1.5 ml EP tube containing 37°C saline. The epididymis was cut into pieces to allow the sperm to fully dissociate in the centrifuge tube, and incubated in a 37°C incubator for 5 minutes. 10 μl of sperm suspension was added to the sperm counting plate, covered with a cover glass, and sperm quality was tested using a computer-assisted semen analysis system. The results are shown in Tables 2 and 3.

[0049] Table 2 Effect of Cuscuta chinensis-Wolfberry Fruit on Sperm Quantity in Rats ( n=7)

[0050]

[0051] Note: Compared with the normal group: 1): P < 0.05, 2): P < 0.01; compared with the model group: 3): P < 0.05, 4): P < 0.01; compared with the L-carnitine group: 5): P < 0.05, 6): P < 0.01; (the same below).

[0052] The results in Table 2 showed that compared with the normal group, the total sperm count and total motile sperm count in the model group were lower than those in the normal group (P < 0.05); compared with the model group, the total sperm count and total motile sperm count in the L-carnitine group, the Cuscuta chinensis-Lycium barbarum granules group, and the Cuscuta chinensis-Lycium barbarum total flavonoids group increased, among which, the differences between the Cuscuta chinensis-Lycium barbarum granules group and the Cuscuta chinensis-Lycium barbarum total flavonoids group and the model group were statistically significant (P < 0.01).

[0053] Table 3 Effects of Cuscuta chinensis-Wolfberry Fruit on the linearity, oscillation and forward motion of rat sperm ( n=7)

[0054]

[0055] The results in Table 3 showed that compared with the normal group, the linearity, oscillation and forward motion of sperm in the model group were significantly lower than those in the normal group (P < 0.01); compared with the model group, the linearity, oscillation and forward motion of sperm in the Cuscuta-Lycium barbarum granules group were increased (P < 0.05).

[0056] 4) HE staining to observe testicular tissue structure and evaluate spermatogenesis function

[0057] After the rats were killed, the testicular tissue was quickly peeled off, the outer membrane of the testis was gently pierced, and the testicles were immediately placed in a testicular tissue fixative, fixed at room temperature for 24 hours, and then transferred to 70% ethanol for storage. After gradient ethanol dehydration, xylene transparency, paraffin embedding and sectioning, dewaxing, staining, differentiation, blueing, gradient alcohol dehydration, staining in eosin staining solution for 5 minutes, dehydration, transparency, neutral gum sealing, and observation of testicular tissue morphology and structure under an optical microscope. The results are shown in Figure 1 At the same time, 10 cross sections of seminiferous tubules were randomly selected from each sample and scored according to the inner diameter of the seminiferous tubules (D), the thickness of the tubular membrane (M), the number of spermatogenic cell layers in the tubular wall (P), and the maturity of spermatogenic cells (S). The total score (TMS) was 20 points. The scoring criteria are shown in Table 4. The results are shown in Table 5.

[0058] Table 4 Testicular spermatogenesis score table

[0059]

[0060] Figure 1 HE staining results showed that the structure of seminiferous tubules in the testicular tissue of rats in the normal group was intact and tightly arranged. Spermatogenic cells at different stages were arranged neatly and orderly, and sperm were visible in the tubular lumen; the walls of the seminiferous tubules in the testicular tissue of the model group were thinned, ruptured, deformed, and loosely arranged. The number of interstitial cells in the testicular tissue decreased, and the spermatogenic cells at all levels were arranged irregularly, with only a small number of sperm in the tubular lumen; after intervention with L-carnitine, Cuscuta-Lycium barbarum granules, and Cuscuta-Lycium barbarum total flavonoids, the morphology of the seminiferous tubules was significantly improved, the arrangement was tighter, the number of spermatogenic cells at all levels increased, the arrangement was orderly, and the number of sperm in the tubular lumen increased.

[0061] Table 5 Effects of Cuscuta chinensis-Lycium barbarum on spermatogenesis in rats ( n=7)

[0062]

[0063] The results in Table 5 showed that compared with the normal group, the spermatogenesis function scores of rats in the model group were significantly decreased (P < 0.01); after treatment, the spermatogenesis function scores of rats in the L-carnitine group, the Cuscuta chinensis-Lycium barbarum granules group, and the Cuscuta chinensis-Lycium barbarum total flavonoids group were significantly improved (P < 0.01).

[0064] 5) Transmission electron microscopy observation of blood-testis barrier structure

[0065] Take 1mm 3 Fresh testicular tissue was placed in electron microscopy fixative and stored at 4°C. After rinsing with 1M phosphate buffer PB (pH 7.4), it was fixed in 1% osmium acid at room temperature in the dark for 2 hours; it was dehydrated in alcohol and acetone gradient, embedded, polymerized, sliced, stained, observed under transmission electron microscopy, and image analysis was collected. Transmission electron microscopy was used to observe the ultrastructure of supporting cells in each group of testicular tissue. The results are shown in Figure 2 .

[0066] Figure 2 The results showed that in the normal group, the tight junctions between supporting cells of testicular tissue were tight and neatly arranged, the mitochondrial morphology was normal, and the mitochondrial cristae were dense; in the model group, the tight junctions were loose and partially broken, the mitochondria were swollen and deformed, the mitochondrial cristae were broken, and some were vacuolated; after intervention, the tight junctions and mitochondrial structure of the Cuscuta chinensis-Lycium barbarum granules group and the Cuscuta chinensis-Lycium barbarum total flavonoids group recovered compared with before.

[0067] 6) ELISA method to detect the levels of SOD, MAD and GSH-Px in testicular tissue

[0068] Weigh 50 mg of testicular tissue and add it to an appropriate amount of physiological saline, grind it to prepare a tissue homogenate, centrifuge it at 3000 rpm for 10 minutes, and take the supernatant. Strictly follow the instructions of the kit and use ELISA to detect the levels of SOD, MAD, and GSH-Px in testicular tissue. The results are shown in Table 6.

[0069] Table 6 Effects of Cuscuta chinensis-Lycium barbarum on the levels of oxidative stress markers in rat testicular tissue ( n=7)

[0070]

[0071] The results in Table 6 show that compared with the normal group, the GSH-PX level in the model group decreased (P>0.05); after drug treatment, the GSH-PX level increased, and the difference between the Cuscuta-Lycium barbarum granules group and the model group was statistically significant (P<0.05). The SOD level in the model group was lower than that in the normal group (P<0.05), and the SOD levels in each group after intervention treatment were higher than those in the model group, and the difference between the Cuscuta-Lycium barbarum total flavonoids group and the model group was statistically significant (P<0.01). The MDA level in the model group was higher than that in the normal group, and the MDA level in the Cuscuta-Lycium barbarum granules group was lower than that in the model group (P>0.05).

[0072] 7) Western blot detection of SIRT1 / Nrf2 pathway and blood-testis barrier-related protein levels in testicular tissue

[0073] Weigh 100 mg of testicular tissue, add 1 ml of RIPA lysis buffer, and homogenize in a cryo-grinding machine at 4 °C and 12000 r / min. -1, centrifuge for 10 minutes and take the supernatant. The protein concentration was measured by BCA method, and the samples were denatured by boiling. The proteins were separated by electrophoresis, transferred to the membrane at a constant current of 400A, and blocked with TBST solution containing 5% skim milk powder at room temperature for 2 hours. After washing the membrane with TBST, the primary antibodies SIRT1 (1:1000), connexin43 (1:1000), Nrf2 (1:1000), ZO-1 (1:5000), occludin (1:25000), and β-catenin (1:25000) were incubated at 4°C overnight. The membrane was washed with TBST 5 times, 7 minutes each time, and the secondary antibody was incubated at room temperature for 2 hours, and developed using ECL luminescent liquid. Image J software was used to determine the gray value of the bands, and the gray ratio of SIRT1, Nrf2 and β-actin, connexin43, ZO-1, occludin, β-catenin and GAPDH reflected the relative expression of the proteins.

[0074] Effects of Cuscuta australis and Lycium barbarum on the expression of SIRT1 / Nrf2 pathway-related proteins in testicular tissue of OAS rats. Figure 3 and Table 7.

[0075] Table 7 Effects of Cuscuta chinensis-Lycium barbarum on the expression levels of SIRT1 and Nrf2 in rat testicular tissue ( n=3)

[0076]

[0077] Figure 3 The results in Table 7 showed that compared with the normal group, the SIRT1 level in the testicular tissue of the model group was decreased (P < 0.01), and the SIRT1 level in the testicular tissue of the Cuscuta chinensis-Lycium barbarum granules group was higher than that of the L-carnitine group (P < 0.05); the Nrf2 level in the model group was significantly lower than that in the normal group (P < 0.01), and the Nrf2 levels in the Cuscuta chinensis-Lycium barbarum granules group and the total flavonoids group were higher than those in the model group (P < 0.05).

[0078] Effects of Cuscuta chinensis and Lycium barbarum on the expression of blood-testis barrier-related proteins in OAS rats. Figure 4 and Table 8.

[0079] Table 8 Effects of Cuscuta chinensis-Lycium barbarum on the levels of blood-testis barrier-related proteins in rat testicular tissue ( n=3)

[0080]

[0081]

[0082] Figure 4The results in Table 8 showed that the occludin protein level in the model group was lower than that in the normal group (P>0.05), and the occludin level in the testicular tissue of the Cuscuta-Lycium barbarum granules group was higher than that in the model group (P<0.01); the ZO-1 protein expression level in the model group was lower than that in the normal group (P<0.05), and the occludin level in the testicular tissue of the Cuscuta-Lycium barbarum granules group was higher than that in the model group and the L-carnitine group (P<0.05); the connexin43 and β-catenin expression levels in the model group were significantly lower than those in the normal group (P<0.01), and the connexin43 level in the testicular tissue of the Cuscuta-Lycium barbarum granules group and the total flavonoids group was higher than that in the model group (P<0.05); the β-catenin expression level in the testicular tissue of the Cuscuta-Lycium barbarum granules group and the total flavonoids group was higher than that in the model group (P>0.05).

[0083] 8) qRT-PCR detection of SIRT1 / Nrf2 pathway and blood-testis barrier-related mRNA levels in testicular tissue

[0084] Weigh 30 mg of testicular tissue and prepare total RNA after lysis, DNA removal, precipitation, protein removal, rinsing, and elution according to the requirements of the total RNA extraction kit. Reverse transcribe the total RNA into cDNA, and use this cDNA as a template to amplify according to the conditions in the instructions. After the reaction is completed, read the CT value on the PCR instrument, and the result is obtained by 2 -△△CT The relative expression of target gene mRNA was calculated by the formula. Primers were provided by Shanghai Shenggong Biotechnology. The primer sequences are shown in Table 9.

[0085] Table 9 Primer sequences

[0086]

[0087] The effects of Cuscuta australis and Lycium barbarum on the expression of SIRT1 / Nrf2 pathway-related mRNA levels in the testicular tissue of OAS rats are shown in Table 10.

[0088] Table 10 Effects of Cuscuta chinensis-Lycium barbarum on SIRT1 and Nrf2 mRNA levels in rat testis tissue ( n=3)

[0089]

[0090] The results in Table 10 showed that compared with the normal group, the SIRT1 and Nrf2 mRNA levels in the model group were significantly decreased (P < 0.01); the SIRT1 and Nrf2 mRNA levels in the L-carnitine group, Cuscuta australis-wolfberry granules, and Cuscuta australis-wolfberry total flavonoids groups were significantly increased compared with the model group (P < 0.01).

[0091] The effects of Cuscuta australis and Lycium barbarum on the expression of blood-testis barrier-related mRNA levels in OAS rats are shown in Table 11.

[0092] Table 11 Effects of Cuscuta australis and Lycium barbarum on the mRNA levels related to the blood-testis barrier in rats ( n=3)

[0093]

[0094] The results in Table 11 showed that the occludin mRNA level in the model group was similar to that in the normal group (P>0.05), and the occludin mRNA levels in each group after drug intervention were higher than those in the model group, among which the differences between the Cuscuta-Lycium barbarum Granules group and the total flavonoids group and the model group were statistically significant (P<0.05). The ZO-1 mRNA level in the model group was lower than that in the normal group (P<0.05), and the ZO-1 mRNA levels in the L-carnitine group and the Cuscuta-Lycium barbarum Granules group were higher than those in the model group (P>0.05). The connexin43 and β-catenin mRNA levels in the model group were lower than those in the normal group (P<0.01), and the connexin43 and β-catenin mRNA levels in the L-carnitine group and the Cuscuta-Lycium barbarum Granules group were higher than those in the model group (P<0.01); the connexin43 mRNA level of the Cuscuta-Lycium barbarum total flavonoids was higher than that in the L-carnitine group (P<0.05).

[0095] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. Application of Cuscuta australis and Lycium barbarum in the preparation of drugs for regulating SIRT1 / Nrf2 signaling pathway to improve oligoasthenospermia.

2. Application of Cuscuta australis and Lycium barbarum in the preparation of drugs for inhibiting blood-testis barrier damage after oxidative stress.

3. Application of Cuscuta australis and Lycium barbarum in the preparation of drugs for regulating SIRT1 / Nrf2 signaling pathway, inhibiting blood-testis barrier damage after oxidative stress, and improving oligoasthenospermia.

4. Application of total flavonoids of Cuscuta australis and Lycium barbarum in the preparation of drugs for regulating SIRT1 / Nrf2 signaling pathway to improve oligoasthenospermia.

5. Application of total flavonoids from Cuscuta australis and Lycium barbarum in the preparation of drugs for inhibiting blood-testis barrier damage after oxidative stress.

6. Application of total flavonoids from Cuscuta seeds and Lycium barbarum in the preparation of drugs that regulate the SIRT1 / Nrf2 signaling pathway, inhibit blood-testis barrier damage after oxidative stress, and improve oligoasthenospermia.

Images