Study on the Mechanism of Radix Astragali against Renal Aging Based on Network Pharmacology.

Radix Astragali is widely used in the traditional Chinese medicine with the effect of antiaging. The purpose of this study is to explore the main active ingredients and targets of Radix Astragali against renal aging by network pharmacology and further to verify the mechanism of the main active ingredients in vitro. TCMSP, ETCM, and TCMID databases were used to screen active ingredients of Radix Astragali. Targets of active ingredients were predicted using BATMAN-TCM and cross validated using kidney aging-related genes obtained from GeneCards and NCBI database. Pathways enrichment and protein-protein interaction (PPI) analysis were performed on core targets. Additionally, a pharmacological network was constructed based on the active ingredients-targets-pathways. HK-2 cell was treated with D-galactose to generate a cell model of senescence. CCK-8 and ß-galactosidase were used to detect the effect of Radix Astragali active components on cell proliferation and aging. ELISA was used to detect the expression of senescence-associated secreted protein (TGF-ß and IL-6) in the cell culture supernatant. Western blot was used to detect the expression of key proteins in the SIRT1/p53 pathway. Five active ingredients (Astragaloside I, II, III, IV and choline) were identified from Radix Astragali, and all these active ingredients target a total of 128 genes. Enrichment analysis showed these genes were implicated in 153 KEGG pathways, including the p53, FoxO, and AMPK pathway. 117 proteins and 572 interactions were found in PPI network. TP53 and SIRT1 were two hub genes in PPI network, which interacted with each other. The pharmacological network showed that the five main active ingredients target on some coincident genes, including TP53 and SIRT1. These targeted genes were involved in the p53, FoxO, and AMPK pathway. Proliferation of HK-2 cells was increased by Astragaloside IV treatment compared with that of the D-Gal treatment group. However, the proliferation of the SA-ß-gal positive cells were inhibited. The expression of TGF-ß and IL-6 in the D-Gal group was higher than that in the normal group, and the treatment of Astragaloside IV could significantly reduce the expression of TGF-ß and IL-6. The expression of SIRT1 in the Astragaloside IV group was higher than that in the D-Gal group. However, the expression of p53 and p21 was less in the Astragaloside IV group than that in the D-Gal group. This study suggested that Astragaloside IV is an important active ingredient of Radix Astragali in the treatment of kidney aging via the SITR1-p53 pathway.

Yishen capsule promotes podocyte autophagy through regulating SIRT1/NF-κB signaling pathway to improve diabetic nephropathy.

Diabetic nephropathy (DN) is a common complication of diabetes. Yishen capsule, composed of Chinese herbs, improves the clinical outcome in DN patients. However, its therapeutic potential and underlying mechanisms require further elucidation. Hence, our study aimed to investigate the underlying mechanisms and therapeutic potential of Yishen capsule in DN. Streptozotocin-induced DN rats were treated with Yishen capsules (1.25 g/kg/day) for 8 weeks. Then, blood glucose and urine protein levels were measured. Hematoxylin and eosin staining and western blot assays were used to examine the histologic changes and gene expression, respectively, in kidney samples. Mouse podocytes were treated with rat serum containing Yishen capsule and transmission electron microscopy was used to examine autophagosome formation. Cell counting kit-8 assay was performed to examine cell proliferation. Western blot and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analyses were conducted to detect changes in gene expression. The localization of SIRT1 was examined in the podocytes using immunocytofluorescence assay. We found that Yishen capsule relieved pathological changes, decreased urine protein, increased SIRT1, LC3-II, and Beclin-1 expression, and reduced acetylated NF-κB p65 expression in vivo. In addition, rat serum containing Yishen capsule showed improved podocyte proliferation, promoted the mRNA and protein levels of LC3-II and Beclin-1, and induced nuclear translocation of SIRT1. Furthermore, it increased SIRT1 expression and decreased mRNA level of NF-κB in the serum. SIRT1 inhibitor increased the mRNA level of NF-κB. Our data suggests that Yishen capsule improves DN by promoting podocyte autophagy via the SIRT1/NF-κB pathway.

Yangjing Capsule Can Improve the Function of the Testicular Angiogenesis through Activating VEGFA/eNOS Signaling Pathway.

BACKGROUND: The testicular microcirculation was an important aspect of testicular physiology and it offered a stable environment for the transport of nutrients and secretary products in the testis. Yangjing capsule (YC), a traditional Chinese compound herbal prescription, has been proved as an effective drug to ameliorate spermatogenesis, promote testosterone synthesis in vivo, and cure spermatogenesis in clinical practice. OBJECTIVE: This study was aimed at understanding the potential mechanisms of YC exerting angiogenic effects in the mouse spermatogenesis dysfunction model induced by cyclophosphamide (CP) and MLTC-1 cells. MATERIALS AND METHODS: Balb/c mice were randomly divided into five groups: control, CP, CP plus YC (630 mg/kg), CP plus YC (1260 mg/kg), and CP plus YC (2520 mg/kg). After 30 days, mice were sacrificed and the expressions of endothelial marker CD34+, angiogenic marker VEGFA, VEGFR1, VEGFR2, and eNOS in the testes of the mice were examined; moreover, Leydig cell line MLTC-1 cells were cultured and treated with different concentrations of YC extracts (YCE), and the expressions of VEGFA, VEGFR1, VEGFR2, and eNOS, as well as the secretion of NO, were evaluated. RESULTS: We observed that YC significantly increased the expressions of VEGFA, VEGFR1, VEGFR2, and eNOS in testes of CP-treated mice; moreover, YCE has led to increased expressions of VEGFA, VEGFR1, VEGFR2, and eNOS and secretion of NO in MLTC-1 in vitro. These data suggested that the YC might be an alternative treatment for the dysfunction of testicular microcirculation by promoting the angiogenesis in the testis.

Current Situation and Reconsideration on the Study of Integrated Chinese and Western Medicine Andrology.

The development of Chinese medicine and Western medicine andrology is based on different social background and academic systems, either Chinese medicine or Western medicine andrology has their limitations, therefore, integration of Chinese and Western medicine (ICWM) andrology is in a great need. After more than 30 years of development, andrology has made great achievements in the construction of specialized academic association, holding academic conferences and publication of academic monographs, and the research progress on this field is mainly in the combination of disease and syndrome, microdifferentiation of symptoms and signs and basic research development. However, the comprehensive theoretic system of ICWM andrology has not yet established, and the related studies are still on the primary stage. In the future studies, great efforts still need to be made to expand the methods for the investigation of ICWM, and make innovations in the field of andrology.

Mechanisms of Yangjing Capsule in Leydig Cell Apoptosis and Testosterone Synthesis via Promoting StAR Expression.

The present study aims to investigate the roles of steroidogenic acute regulatory protein (StAR) in Yangjing Capsule (YC) induced anti-apoptotic effects on Leydig cells and the related mechanism. Leydig tumor cells (MLTC-1) were cultured and treated with YC, and immunofluorescence assay was performed to examine the expression of StAR; furthermore, luciferase reporter assay was conducted to evaluate the impact of YC on StAR promoter; next, MLTC-1 cells were treated with StAR small interfering RNA (siRNA), and flow cytometry was carried out to examine the effect of StAR siRNA on the apoptosis of the cells; furthermore, quantitative (q)RT-PCR and Western blot methods was used to determine the expression of StAR and apoptosis related molecules Bcl-2, Bax and Caspase-3 on both mRNA and protein levels in different groups; finally the secretion of testosterone in different groups was examined by radioimmunoassay. We observed that the YC can increase the expression of StAR in a dose-dependent manner, and YC can activate the promoter of StAR; moreover, transfection of StAR siRNA can block YC induced anti-apoptotic effects and increased production of testosterone. In conclusion, our results suggested that YC might suppress the apoptosis of MLTC-1 cells and enhance the production of testosterone through regulating the expression of StAR.

Yangjing Capsule extract promotes proliferation of GC-1 spg cells via up-regulated POU3F1 pathway.

As is similar to glial cell line-derived neurotrophic factor (GDNF), the Yangjing Capsule (YC) extract could also lead to proliferation of spermatogonial stem cells (SSCs) by stimulating the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) pathway; however, the regulatory effect of YC extract on the expression of POU3F1 still remains unknown. The objective of this study is to determine whether the transcription factor POU3F1 is up-regulated by YC extract through the PI3K/AKT signaling pathway to regulate SSCs survival and proliferation. Cultured GC-1 spermatogonial (spg) cells were treated with 0.01, 0.1, and 1 mg/mL YC extract for 48 h. Cell viability was analyzed using MTT assay, while POU3F1 expression was quantitatively detected using real time-polymerase chain reaction and Western blot analysis. POU3F1, GDNF family receptor alpha1 (GFRα1) short interfering ribonucleic acid (siRNA), and LY294002 (PI3K inhibitor) were applied as blockers to explore the underlying pathway. After 48 h treatment with YC extract, GC-1 spg cells proliferated and POU3F1 expression was significantly increased in a dose-dependent manner. POU3F1 siRNA partially blocked those effects of YC extract. Both GFRα1 siRNA and LY294002, as upstream blockers, reduced POU3F1 expression induced by YC extract. The conclusion is that YC extract promotes proliferation of GC-1 spg cells via up-regulation of POU3F1. The potential mechanism is that YC extract triggers the activation of the PI3K/AKT pathway and then up-regulates POU3F1 expression.

[Bushen Daozhuo Granules for type â ¢ prostatitis: A multicenter randomized controlled clinical trial].

OBJECTIVE: To study the safety and efficacy of Bushen Daozhuo Granules (BDG) in the treatment of type Ⅲ prostatitis. METHODS: This multicenter randomized controlled clinical trial included 478 patients with type Ⅲ prostatitis, 290 in the trial group and 188 as controls, the former treated with BDG at 200 ml bid and the latter with tamsulosin hydrochloride sustainedrelease capsules at 0.2 mg qd, both for 4 weeks. Before treatment, after 4 weeks of medication, and at 4 weeks after drug withdrawal, we obtained the NIH Chronic Prostatitis Symptom Index (NIHCPSI) scores and compared the safety and effectiveness rate between the two groups of patients. RESULTS: Compared with the baseline, the NIHCPSI score was markedly decreased in the control group after 4 weeks of medication (21.42 ± 4.02 vs 15.67 ± 3.65, P < 0.05) but showed no statistically significant difference from that at 4 weeks after drug withdrawal (19.03 ± 3.86) (P>0.05), while the NIHCPSI score in the trial group was remarkably lower than the baseline both after 4 weeks of medication and at 4 weeks after drug withdrawal (10.92 ± 2.06 and 12.91 ± 2.64 vs 21.58 ± 3.67, P < 0.05). The trial group exhibited both a higher rate of total effectiveness and safety than the control (P < 0.05). CONCLUSIONS: BDG is safe and effective for the treatment of type Ⅲ prostatitis.

[Explanation of 'Essence and Blood from the Same Source' in Theory of Chinese Medicine from the Roles of Vascular Endothelial Growth Factor in Spermatogenesis].

'Essence and blood from the same source', as one of basic theories of Chinese medi- cine (CM), is important for syndrome typing and clinical medication. Vascular endothelial growth factor (VEGF) can be produced and secreted in Leydig cells and Sertoli cells, which can not only promote the formation of new blood vessels, but also regulate synthesis of testosterone and the proliferation and dif- ferentiation of germ cells. The roles of VEGF in the spermatogenesis reveal scientific connotations of 'essence and blood from the same source', and it plays an important role in guiding clinical practice of CM.

The Stimulative Effect of Yangjing Capsule on Testosterone Synthesis through Nur77 Pathway in Leydig Cells.

Yangjing Capsule (YC), an innovative Chinese medicine based on traditional prescription, promotes testosterone synthesis by upregulating the expression of steroidogenic enzymes. Nur77 as a nuclear receptor is known to regulate the expression of many steroid synthetases. This study aimed to explore the potential mechanisms by which YC regulates testosterone synthesis in Leydig cells. Real-time PCR and Western blot analysis were employed to assess the expressions of steroidogenic enzymes and Nur77 after treating MLTC-1 cells with YC. The luciferase reporter gene assay was performed to detect the activity of Nur77 gene promoter. Also, the expressions of steroid synthases were detected after Nur77 gene was knocked down. YC significantly stimulated Nur77 production and upregulated StAR and HSD3B expression, and this agrees with the activity of Nur77 gene promoter that was significantly enhanced by YC. Interestingly, knockdown of Nur77 blocked the above YC's effects and consequently inhibited testosterone synthesis in MLTC-1 cells. YC promotes StAR and HSD3B expression and upregulates testosterone synthesis in Leydig cells, which is mediated by Nur77 pathway.

Yangjing Capsule Ameliorates Spermatogenesis in Male Mice Exposed to Cyclophosphamide.

Yangjing capsule (YC), a traditional Chinese compound herbal preparation, has been proven as an effective drug to improve spermatogenesis in clinical practice. However, its pharmacological mechanisms were not fully clarified. This study was designed to investigate the protective effects of YC on spermatogenesis in the mouse model of spermatogenesis dysfunction induced by cyclophosphamide (CP). The administration of YC significantly increased the epididymal index, sperm count, and sperm motility of model mice. Histopathological changes demonstrated that CP caused obvious structural damage to testis, which were reversed by the administration of YC. Results from TUNEL assay showed that treatment with YC dramatically decreased the apoptosis of spermatogenic cell induced by CP. Moreover, YC treatment could inhibit the mRNA and protein expression of Bax to Bcl-2 and also raised expression of AR at both mRNA and protein levels. These data suggest that YC might ameliorate spermatogenesis in male mice exposed to CP through inhibiting the apoptosis of spermatogenic cell and enhancing the actions of testosterone in spermatogenesis.

Corrigendum to "Yangjing Capsule Extract Promotes Proliferation of GC-1 Spg Cells".

[This corrects the article DOI: 10.1155/2014/640857.].

Yangjing Capsule Extract Promotes Proliferation of GC-1 Spg Cells.

Objective. To investigate the effect of Yangjing Capsule (YC) extract on proliferation of GC-1 spermatogonia (spg) cells and the mechanism. Methods. GC-1 spg cells were treated with 0.01, 0.1, and 1 mg/mL YC extract. MTT assay was performed to detect the cell viability. Flow cytometry was used to measure the cell cycle and apoptosis of GC-1 spg cells. Real-time PCR and western blot were applied to determine the mRNA and protein expression of Oct-4 and Plzf. Gfr α 1 knockdown and LY294002 (PI3K inhibitor) were applied to explore the underlying mechanism. Results. After 48 h treatment of YC, the viability of GC-1 spg cells increased significantly and the ratio of apoptotic cells reduced significantly. The increased mRNA and protein expression of Oct-4 and Plzf suggested YC promoted self-renewal of GC-1 spg cells. Both Gfr α 1 siRNAs and LY294002 treatments held back YC extract's stimulation effects on mRNA and protein expression of Oct-4 and Plzf and consequently inhibited the proliferation of GC-1 spg cells induced by YC extract. Conclusion. YC extract could stimulate the proliferation of GC-1 spg cells. Partly via Gfr α 1, YC extract is able to trigger the activation of PI3K pathway and finally lead to self-renewal of GC-1 spg cells.

[Effects of Yangjing capsule on the ultrastructure of seminal vesicles in aged rats].

OBJECTIVE: To observe the effects of Yangjing Capsule (YJC) on the ultrastructure of seminal vesicles in aged male rats, and explore its mechanism of improving the secretion of seminal vesicles. METHODS: Fifty male SD rats aged 18 -20 months were randomly and equally divided into a control group, a testosterone undecanoate group, and a high-dose, a medium-dose and a low-dose YJC group, all fed intragastrically for 30 days. Then the seminal vesicles of the rats were removed and the seminal fluid squeezed into the test tube to be weighed and measured for the concentration of seminal vesicle fluid fructose, and the bilateral seminal vesicles were placed in formaldehyde and glutaraldehyde fixatives for histological observation. RESULTS: The seminal vesicle gland viscera coefficient, seminal vesicle fluid weight and fructose concentration of the rats were (1164.5 +/- 212.6) g/g x 10(6), (0.83 +/- 0.30) g and (4.35 +/- 0.31) mg/ml in the control group, (1510.5 +/- 313.1) g/g x 10(6), (0.82 +/- 0.25) g and (5.35 +/- 0.71) mg/ml in the testosterone undecanoate group, (1484.3 +/- 262.7) g/g x 10(6), (1.14 +/- 0.18) g and (5.30 +/- 0.45) mg/ml in the high-dose YJC group, (1396.6 +/- 268.9) g/g x 10(6), (0.83 +/- 0.24) g and (4.71 +/- 0.41) mg/ml in the medium-dose YJC group, and (1475.0 +/- 305.2) g/g x 10(6), (0.74 +/- 0.28) g and (4.50 +/- 0.23) mg/ml in the low-dose YJC group. Compared with the control, high-dose YJC significantly improved seminal vesicle secretion (P < 0.05), while medium- and low-dose only achieved a trend of improvement. After HE staining, the YJC groups showed more active epithelial hyperplasia, increased cellular layers, rich and transparent cytoplasm with abundant secretory granules, fat droplets and lipofuscin, blurred glandular cavity edge, and eosinophilic intraluminal secretions, as compared with the control group. The structural change was most significant in the high-dose group. Statistically significant differences were observed in the numerical density and bulk density of the secretory granules between the YJC and control groups (P < 0.05). CONCLUSION: Yangjing Capsule can improve the secretion of seminal vesicles by increasing the secretory granules of the main

[Regulatory effect of Yangjing Capsule on the expressions of phosphor-Erk1 and phosphor-Akt1 in the corpus cavernosum of aged rats].

OBJECTIVE: To observe the effect of Yangjing Capsule on the expressions of P-Erk1 and P-Akt1 in the corpus cavernosum of aged rats and to explore its action mechanism. METHODS: Forty 24-month-old SD rats were equally randomized into an experimental and a control group, 10 selected from each group for detection of the eNOS expression before medication. The rats in the experimental group (n = 10) were treated intragastrically with Yangjing Capsule, while those in the control with physiological saline, both at 0.5 g per kg body weight per day for 4 weeks. Then all the rats were killed, and the corpus cavernosum tissues harvested for determination of the expressions of eNOS, P-Erk1 and P-Akt1 by HE staining, RT-PCR and immunohistochemistry. RESULTS: Obvious distension was observed in the experimental group, but not in the control. The expression of eNOS in the rat corpus cavernosum was significantly higher after treatment than before treatment in the experimental group (24.10 +/- 2.40 vs 18.80 +/- 2.04, P < 0.05), but presented no obvious difference in the control (18.15 +/- 1.98 vs 19.35 +/- 1.50, P > 0.05), and it was remarkably higher in the former than in the latter (P < 0.05). The expression of P-Erk1 was markedly lower in the experimental than in the control rats (0.71 +/- 0.13 vs 0.83 +/- 0.17, P < 0.01), but that of P-Akt1 exhibited no remarkable difference in the same condition of GAPDH between the two groups (0.58 +/- 0.17 vs 0.48 + 0.13, P > 0.01). CONCLUSION: Both P-Erk1 and P-Akt were expressed in the corpus cavernosum of the aged rats. Yangjing Capsule could improve the sexual function of aged rats, which might be associated with the lowered expression of P-Erk1.

Effects of the yangjing capsule extract on steroidogenesis and apoptosis in mouse leydig cells.

Objectives. This study aimed to explore the effect and mechanism of Yangjing capsule on testosterone secretion in mouse Leydig tumor cells (MLTC-1). Methods. MLTC-1 cells were treated with the Yangjing capsule extract for 24 h. The testosterone level in medium was measured by radioimmunoassay. The expression of steroidogenic enzymes (StAR, CYP11A1, and HSD3B) in the cells was examined using real-time RT-PCR and immunoblotting. Additionally, MLTC-1 cells were treated for 48 h in a serum-free medium. The cell viability was measured by MTT assay. The cell cycle and apoptosis were analyzed using flow cytometry. The expression of activated caspase-3 was analyzed using RT-PCR and a colorimetric protease assay. Results. The Yangjing capsule extract increased testosterone production and the expression of StAR, CYP11A1, and HSD3B mRNAs and proteins compared with the control. H89 significantly inhibited these effects. The medicine improved the viability of MLTC-1 cells, decreased the number of cells in G0/G1 phase, and increased the number of cells in S-phase, as well as prevented cell apoptosis by inhibiting caspase-3. Conclusion. The Yangjing capsule can stimulate MLTC-1 cells to secrete testosterone and may be an alternative treatment for diseases characterized by insufficient testosterone production.

[Effects of Yixin Kangtai capsule on spermatogenesis and the levels of superoxide dismutase and malondialdehyde in aged SD rats].

OBJECTIVE: To observe the effects of Yixin Kangtai Capsule (YKC) on sperm concentration and motility, superoxide dismutase (SOD) and malondialdehyde (MDA) in aged SD rats, and to explore its possible mechanisms of action. METHODS: We randomly assigned fifty 24-month-old male SD rats to five groups of equal number: blank control, testosterone undecanoate, high-dose YKC, medium-dose YKC and low-dose YKC, and treated them intragastrically with physiological saline at 10 ml/kg, testosterone un-decanoate suspension at 4 mg/kg, and YKC at 0.6 g/kg, 0.3 g/kg and 0.15 g/kg, respectively, once a day for 30 days. Then we harvested the epididymides, which were stripped of fat and envelope, cut into pieces and placed in the saline at 37 degrees C for 10 to 15 minutes. Then we detected the sperm parameters with the Weili CASA sperm analysis system, measured the levels of serum SOD and MDA, and, following HE staining, observed the histomorphological changes of the testicular tissue under the light microscope. RESULTS: Compared with the controls, the rats treated with high-, medium- and low-dose YKC exhibited significantly increased sperm concentration ([152.3 +/- 24.0] x 10(6)/ml vs [334.5 +/- 97.7], [302.7 +/- 158.9] and [221.4 +/- 75.6] x 10(6)/ml, P < 0.05) and markedly improved sperm motility ([26.5 +/- 2.0]% vs [35.5 +/- 5.9], [33.1 +/- 5.4] and [28.4 +/- 2.1]%, P < 0.01). High-dose YKC significantly increased the level of serum SOD ([22.0 +/- 5.3] U/ml, P < 0.01) and reduced the content of MDA ([13.6 +/- 4.6] nmol/ ml, P < 0.01). Testicular histology showed that YKC induced more active proliferation of cells, and increased the number of dividing cells and that of mature sperm in the lumen, with little interstitium, abundant blood vessels, and well-developed stromal cells. CONCLUSION: Yixin Kangtai Capsule can improve spermatogenesis in aged SD rats by regulating the levels of SOD and MDA.

[Yangjing capsule plus low-dose tadalafil for functional anejaculation].

OBJECTIVE: To search for an effective therapy for functional anejaculation (AE). METHODS: Fifty-five AE patients were randomized into a treatment group (n = 30) and a control group (n = 25), the former treated with Yangjing Capsule (once 5 pills, tid) and low-dose tadalafil (5 mg, qd alt, 1 h before bedtime), while the latter with oral ephedrine (25 mg before bedtime). Meanwhile, the patients were advised to expose themselves to sexual stimulation, reduce the frequency of sexual intercourses and quit masturbation. The medication lasted 1-3 months, followed by observation of the therapeutic effects. RESULTS: The total effectiveness rate was 83.34% in the treatment group, 11 cases cured, 8 obviously improved, 6 improved and 5 unimproved, significantly higher than 40.00% in the control group, 4 cases cured, 3 obviously improved, 3 improved and 15 unimproved (P < 0.05). CONCLUSION: Yangiing Capsule plus low-dose tadalafil is safe and effective for the treatment of functional anejaculation.