In vivo and in vitro protective effects of the Wuzi Yanzong pill against experimental spermatogenesis disorder by promoting germ cell proliferation and suppressing apoptosis.

ETHNOPHARMACOLOGICAL RELEVANCE: Wuzi Yanzong pill (WZYZP) is a classical traditional Chinese medicine (TCM) formula originated from the Tang dynasty. WZYZP has a long history of use for reinforcing kidney and alleviating male infertility in China. AIM OF THE STUDY: The effect of WZYZP on male infertility and the mechanism underlying this effect was not clarified clearly. Therefore, this study aimed to investigate the protective effect of WZYZP in experimental spermatogenesis disorder via in vivo and in vitro studies, to promote the use of this formula for the treatment of spermatogenesis disorder. MATERIAL AND METHODS: Male SD rats were exposed to tripterygium glycosides to induce experimental spermatogenesis disorder, and WZYZP was subsequently administrated at different dosages for treatment. Sperm counts, sperm motility, and serum hormone levels were detected. HE staining and TUNEL staining were performed to evaluate the pathological lesions and apoptosis of testes, respectively. Next, germ cells were isolated from spermatogenesis disorder-model rats and treated with WZYZP- containing serum at different concentrations. CCK-8 assay and flow cytometry assay were performed to detect cell proliferation and apoptosis. Immunofluorescence assay, qRT-PCR and Western blotting analyses were performed to detect the expression of Beclin 1, LC3 and TGF-ß-PI3k/AKT-mTOR pathway - related factors, including TGF-ß, PI3K, AKT, mTOR, 4 EBP-1 and p70S6K. RESULTS: In vivo experiments showed that WZYZP protected against spermatogenesis disorder in model rats by improving sperm count and motility, as well as restoring serum hormone levels. HE and TUNEL staining demonstrated that the pathological injuries and cell apoptosis in testes of the model rats were alleviated by WZYZP treatment. Moreover, in vitro experiments of germ cells isolated from spermatogenesis disorder-model rats showed that WZYZP treatment increased the cell proliferation, inhibited cell apoptosis and autophagy. qRT-PCR and Western blotting assay results showed that this protective effect was associated with the regulation of the TGF-ß/PI3K/AKT/mTOR signaling pathway. The expression levels of p-PI3K/PI3K, p-AKT/AKT, p-mTOR/mTOR, 4 EBP-1 and p70S6K were increased, while TGF-ß was inhibited in the WZYZP treated groups. CONCLUSION: The results showed that WZYZP could protect against experimental spermatogenesis disorder by increasing the germ cell proliferation and inhibiting their apoptosis. Our support the clinical use of this formula for the management of spermatogenesis disorder.

Efficacy and Safety of Chuanxiong Qingnao Granule in Patients with Migraine: A Randomized, Double-Blind, Placebo-Controlled Trial.

OBJECTIVE: To evaluate the efficacy and safety of Chuanxiong Qingnao Granule (CQG) to treat migraine. METHOD: This study was a randomized, double-blind, placebo-controlled trial. All migraineurs were recruited and randomly assigned into a treatment group treated with CQG and a control group treated with a placebo. The whole research process included a 4-week baseline, 12-week intervention, and 12-week follow-up. The primary outcome was responder rate, defined as the percentage of migraineurs with 50% or more reduction in the frequency of migraine attack during treatment and posttreatment period compared with the baseline. The secondary outcomes were the number of migraine days, migraine attack frequency, visual analogue scale (VAS), Fatigue Severity Scale (FSS), Hamilton Depression Scale (HAMD), and Migraine Disability Assessment (MIDAS). RESULTS: A total of 346 migraineurs completed the research and were included in the intention-treatment analyses. The response rates differed significantly between the treatment group and the control group (71.5% vs. 12.1% at week 12 and 83.1% vs. 3.4% at week 24). Attack frequency, days of headache attack, VAS, FSS, HAMD, and MIDAS decreased at week 12 in both groups with more reduction in the treatment group (P < 0.001). No severe adverse events were observed in this trial. CONCLUSION: Chuanxiong Qingnao Granule can significantly improve headache symptoms in patients with migraine while improving disability, fatigue, and depression with a good safety profile.

Uncovering the mechanisms of leech and centipede granules in the treatment of diabetes mellitus-induced erectile dysfunction utilising network pharmacology.

ETHNOPHARMACOLOGICAL RELEVANCE: Diabetes mellitus-induced erectile dysfunction (DMED) is one of the most common complications of diabetes mellitus. Leech and centipede granules (LCG) have traditionally been used as blood-activating agents in various ethnomedicinal systems of East Asia, especially in China. It is often used to regulate bodily functions and considered as adjuvant therapy for promoting blood circulation, alleviating blood coagulation, activating meridians, and relieving stasis. AIM OF THE STUDY: This study aimed to identify potential genes and mechanisms of LCG on DMED from the network pharmacological perspective. MATERIALS AND METHODS: The active components of LCG were identified by UHPLC-Q-TOF-MS, TCMID, and the BATMAN-TCM databases, and the disease targets of DMED were obtained from the DisGeNET, CooLGeN, GeneCards databases. After identifying DMED targets of LCG, a protein-protein interaction (PPI) network was constructed. Hub genes and significant modules were identified via the MCODE plug-in of Cytoscape software. Then, significant signaling pathways of the modules were identified using the Metascape database. The probable interaction mode of compounds-hub genes is examined using Molecular Operating Environment (MOE) docking software. Besides, we investigated the effects and mechanisms of LCG on improving erectile function in the streptozotocin (STZ)-induced diabetic rats model. RESULTS: Combined UHPLC-Q-TOF-MS analysis with network pharmacology study, 18 active compounds were selected for target prediction. There are 97 common target genes between LCG and DMED. Enrichment of the KEGG pathway mainly involves in the calcium signaling pathway, NF-kappa B signaling pathway, cGMP-PKG signaling pathway, HIF-1 signaling pathway, PI3K-Akt signaling pathway, and mTOR signaling pathway. Nine hub genes were regulated by LCG in DMED, including CXCL8, NOS3, CRH, TH, BDNF, DRD4, ACE, CNR1, and HTR1A. The results of molecular docking analysis showed that the tyrosin, ursolic acid, and L-Histidine has a relatively stable interaction with corresponding hub genes via generating hydrogen bonds, H-π, and π-π interactions. Significantly, the results in docking predicted a higher affinity of vardenafil to the hub genes compared to the tyrosin, ursolic acid, and L-Histidine. Furthermore, LCG increased the testosterone, erection frequency, the ratio of ICP and MAP, SOD, cGMP, cAMP as well as decreased the MDA, and AGEs expression levels. And, LCG ameliorated the histological change of penile tissues in DMED rats. Hence, LCG attenuates oxidative stress, increases NO production; For the mechanism exploration, LCG could significantly upregulate the mRNA and protein expression of CNR1, NOS3, CRH, TH, BDNF, and DRD4, whereas CXCL8, ACE, and HTR1A levels were significantly higher than those in the DMED group. Moreover, LCG activates the NO/cGMP/PKG pathway, PI3K/Akt/nNOS pathway, cAMP/PKA pathway, and inhibits the HIF-1α/mTOR pathway to improve erectile function. CONCLUSIONS: Our results suggest that LCG maybe offer a new therapeutic basis for the treatment of DMED via altering the gene expression of involved metabolic pathways.

Wuzi Yanzong Pill-Based on Network Pharmacology and In Vivo Evidence-Protects Against Spermatogenesis Disorder via the Regulation of the Apoptosis Pathway.

The crisis of male infertility is an issue of human reproductive health worldwide. The Wuzi Yanzong pill (WZYZP) is a traditional Chinese medicine prescription that shows efficacy in kidney reinforcement and essence benefit to ameliorate male reproductive dysfunctions. However, the pharmacological mechanisms of the WZYZP on male infertility have not been investigated and clarified clearly. This study was designed to investigate the effects of the WZYZP on spermatogenesis disorder and explore its underlying pharmacological mechanisms. First, based on a network pharmacology study, 39 bioactive compounds and 40 targets of the WZYZP associated with spermatogenesis disorder were obtained, forming a tight compound-target network. Molecular docking tests showed tight docking of these compounds with predicted targeted proteins. The protein-protein interaction (PPI) network identified TP53, TNF, AKT1, Bcl-XL, Bcl-2, and IκBA as hub targets. The Kyoto Encyclopedia of Genes and Genomes pathway network and pathway-target-compound network revealed that the apoptosis pathway was enriched by multiple signaling pathways and multiple targets, including the hub targets. Subsequently, the chemical characterization of WZYZP was analyzed using liquid chromatography to quadrupole/time-of-flight mass spectrometry, and 40 compounds in positive ion mode and 41 compounds in negative ion mode in the WZYZP were identified. Furthermore, based on the prediction of a network pharmacology study, a rat model of spermatogenesis disorder was established to evaluate the curative role and underlying mechanisms of the WZYZP. The results showed that WZYZP treatment improved rat sperm quality and attenuated serum hormone levels, reversed histopathological damage of the testis, reduced cell apoptosis in testis tissues, and ameliorated the expression of the predicted hub targets (TP53, TNF-α, AKT1, NFκB, and IκBA) and the apoptosis related proteins (Bcl-XL, Bcl-2, Bax, Caspase 3, and Caspase 9). These results indicated that the WZYZP has a protective effect on spermatogenesis disorder, suggesting that it could be an alternative choice for male infertility therapy.

Protection by salidroside against bone loss via inhibition of oxidative stress and bone-resorbing mediators.

Oxidative stress is a pivotal pathogenic factor for bone loss in mouse model. Salidroside, a phenylpropanoid glycoside extracted from Rhodiola rosea L, exhibits potent antioxidative effects. In the present study, we used an in vitro oxidative stress model induced by hydrogen peroxide (H(2)O(2)) in MC3T3-E1 cells and a murine ovariectomized (OVX) osteoporosis model to investigate the protective effects of salidroside on bone loss and the related mechanisms. We demonstrated that salidroside caused a significant (P<0.05) elevation of cell survival, alkaline phosphatase (ALP) staining and activity, calcium deposition, and the transcriptional expression of Alp, Col1a1 and Osteocalcin (Ocn) in the presence of H(2)O(2). Moreover, salidroside decreased the production of intracellular reactive oxygen species (ROS), and osteoclast differentiation inducing factors such as receptor activator of nuclear factor-kB ligand (RANKL) and IL-6 induced by H(2)O(2). In vivo studies further demonstrated that salidroside supplementation for 3 months caused a decrease in malondialdehyde (MDA) and an increase in reduced glutathione (GSH) concentration in blood of ovariectomized mouse (P<0.05), it also improved trabecular bone microarchitecture and bone mineral density in the fourth lumbar vertebra and distal femur. Our study indicated that the protection provided by salidroside in alleviating bone loss was mediated, at least in part, via inhibition of the release of bone-resorbing mediators and oxidative damage to bone-forming cells, suggesting that salidroside can be used as an effective remedy in the treatment or prevention of osteoporosis.