The effect of semen cuscutae flavonoid on Sertoli cells and blood-testis barrier in male infertility: integrating network pharmacology and experimental verification.

CONTEXT: Semen cuscutae is commonly used to treat male infertility (MI), and semen cuscutae flavonoid (SCF) is the main active component of semen cuscutae. The therapeutic mechanism of SCF on MI is still unclear. OBJECTIVE: To clarify the mechanisms of SCF against MI. MATERIALS AND METHODS: Network pharmacology and molecular docking were used to predict the potential pathways of SCF against MI. Primary Sertoli cells (SCs) were extracted from testis of 60-day-old rats and divided into Control, Model, and 3 treatment groups. The Control and Model groups were given normal medium, the treatment groups were treated with various concentrations of SCF-containing medium (200, 400, and 800 µg/mL). After 24 h, the Model and treatment groups were exposed to heat stress at 43 °C for 15 min. Western blotting and immunohistochemistry were used to detect the expression of targets. RESULT: Network pharmacology indicated that the treatment of SCF on MI was closely related to PI3K-AKT signaling pathway. The in vitro experiments showed that SCF could up-regulated the expression of AKT, AR, occludin, and Ki67, and down-regulated the expression of CK-18 in SCs after heat stress. The AKT inhibitor could block this process. CONCLUSIONS: SCF can treat MI by regulating the proliferation and differentiation of SCs and the integrity of the blood-testis barrier. The study could provide experimental basis for clinical research.

Deciphering the Mechanism of Wogonin, a Natural Flavonoid, on the Proliferation of Pulmonary Arterial Smooth Muscle Cells by Integrating Network Pharmacology and In Vitro Validation.

Wogonin is one of the main active components of Scutellaria baicalensis, which has anti-inflammatory, anti-angiogenesis, and anti-fibrosis effects. Nevertheless, the effect of wogonin on pulmonary hypertension (PH) still lacks systematic research. This study aims to elucidate the potential mechanism of wogonin against PH through network pharmacology and further verify it through biological experiments in pulmonary arterial smooth muscle cells (PASMCs). The potential targets and pathways of wogonin against PH were predicted and analyzed by network pharmacology methods and molecular docking technology. Subsequently, the proliferation of PASMCs was induced by platelet-derived growth factor-BB (PDGF-BB). Cell viability and migration ability were examined. The method of Western blot was adopted to analyze the changes in related signaling pathways. Forty potential targets related to the effect of wogonin against PH were obtained. Based on the protein-protein interaction (PPI) network, gene-ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) enrichment, and molecular docking, it was shown that the effect of wogonin against PH is closely related to the proliferation of PASMCs and the hypoxia-inducible factor-1α (HIF-1α) pathway. A variety of results from biological experiments verified that wogonin can effectively inhibit the proliferation, migration, and phenotypic transformation of PDGF-BB-mediated PASMCs. In addition, the anti-proliferation effect of wogonin may be achieved by regulating HIF-1/ NADPH oxidase 4 (NOX4) pathway.

Probing the Potential Mechanism of Quercetin and Kaempferol against Heat Stress-Induced Sertoli Cell Injury: Through Integrating Network Pharmacology and Experimental Validation.

Quercetin and kaempferol are flavonoids widely present in fruits, vegetables, and medicinal plants. They have attracted much attention due to their antioxidant, anti-inflammatory, anticancer, antibacterial, and neuroprotective properties. As the guarantee cells in direct contact with germ cells, Sertoli cells exert the role of support, nutrition, and protection in spermatogenesis. In the current study, network pharmacology was used to explore the targets and signaling pathways of quercetin and kaempferol in treating spermatogenic disorders. In vitro experiments were integrated to verify the results of quercetin and kaempferol against heat stress-induced Sertoli cell injury. The online platform was used to analyze the GO biological pathway and KEGG pathway. The results of the network pharmacology showed that quercetin and kaempferol intervention in spermatogenesis disorders were mostly targeting the oxidative response to oxidative stress, the ROS metabolic process and the NFκB pathway. The results of the cell experiment showed that Quercetin and kaempferol can prevent the decline of cell viability induced by heat stress, reduce the expression levels of HSP70 and ROS in Sertoli cells, reduce p-NF-κB-p65 and p-IκB levels, up-regulate the expression of occludin, vimentin and F-actin in Sertoli cells, and protect cell structure. Our research is the first to demonstrate that quercetin and kaempferol may exert effects in resisting the injury of cell viability and structure under heat stress.

Mechanistic and therapeutic perspectives of baicalin and baicalein on pulmonary hypertension: A comprehensive review.

Pulmonary hypertension (PH) is a chronic and fatal disease, for which new therapeutic drugs and approaches are needed urgently. Baicalein and baicalin, the active compounds of the traditional Chinese medicine, Scutellaria baicalensis Georgi, exhibit a wide range of pharmacological activities. Numerous studies involving in vitro and in vivo models of PH have revealed that the treatment with baicalin and baicalein may be effective. This review summarizes the potential mechanisms driving the beneficial effects of baicalin and baicalein treatment on PH, including anti-inflammatory response, inhibition of pulmonary smooth muscle cell proliferation and endothelial-to-mesenchymal transformation, stabilization of the extracellular matrix, and mitigation of oxidative stress. The pharmacokinetics of these compounds have also been reviewed. The therapeutic potential of baicalin and baicalein warrants their continued study as natural treatments for PH.

Wuzi-Yanzong prescription alleviates spermatogenesis disorder induced by heat stress dependent on Akt, NF-κB signaling pathway.

Akt and nuclear factor kappa B (NF-κB) signaling pathways are involved in germ cell apoptosis and inflammation after testicular heat stress (THS). We observed that after THS induced by the exposure of rat testes to 43 °C for 20 min, their weight decreased, the fraction of apoptotic testicular germ cells significantly increased, and the proliferation of germ cells was inhibited. In addition, THS lowered serum testosterone (T) level, whereas the levels of follicle stimulating hormone and luteinizing hormone were not significantly changed. The ultrastructure of the seminiferous tubules became abnormal after THS, the structure of the blood-testis barrier (BTB) became loose, and the Sertoli cells showed a trend of differentiation. The level of phosphorylated Akt was reduced, whereas the amount of phosphorylated NF-κB p65 was augmented by THS. Wuzi-Yanzong (WZYZ), a classic Chinese medicine prescription for the treatment of male reproductive dysfunctions, alleviated the changes induced by THS. In order to determine the mechanism of action of WZYZ, we investigated how this preparation modulated the levels of T, androgen receptor (AR), erythropoietin (EPO), EPO receptor, and Tyro-3, Axl, and Mer (TAM) family of tyrosine kinase receptors. We found that WZYZ activated the Akt pathway, inhibited the Toll-like receptor/MyD88/NF-κB pathway, and repaired the structure of BTB by regulating the levels of T, AR, TAM receptors, and EPO. In conclusion, these results suggest that WZYZ activates the Akt pathway and inhibits the NF-κB pathway by acting on the upstream regulators, thereby improving spermatogenesis deficit induced by THS.

Effect of Wuzi Yanzong Pills on Sertoli cells and blood-testis barrier in heat-stressed rats based on Akt signalling pathway.

The blood-testis barrier (BTB) of Sertoli cells (SCs) is an important biological barrier that maintains spermatogenesis and provides a favourable microenvironment for spermatogenesis. However, heat stress can directly damage the BTB structural proteins of testicular SCs, leading to dyszoospermia. Wuzi Yanzong Pills (WYP) is a traditional Chinese medicine formula used to treat male reproductive diseases. However, whether WYP could ameliorate heat stress injury in primary SCs extracted from rat testes and BTB proteins remains unknown. Here, treatment with WYP (low, medium and high dose) increased the SC viability and the proliferation of cell antigen Ki67 significantly. Additionally, it promoted SC maturation, which presented in the form of increased androgen receptors (ARs) and decreased cytokeratin 18 (CK-18) in three WYP dose groups. WYP upregulated BTB proteins such as zonula occludens 1 (ZO-1) and occludin across all WYP groups and decreased phosphorylated Akt (p-Akt) in the middle and high-dose groups; however, ZO-1 and occludin recovery were reduced with the presence of Akt inhibitor in WYP groups. WYP improved SC viability and proliferation, and ameliorated dedifferentiation and BTB-proteins damaged by heat stress via Akt signalling. The findings present theoretical support for the effects of WYP in the management of dyszoospermia and male infertility.

Lycium barbarum Polysaccharide Ameliorates Heat-Stress-Induced Impairment of Primary Sertoli Cells and the Blood-Testis Barrier in Rat via Androgen Receptor and Akt Phosphorylation.

Male infertility induced by heat stress has been attracting more and more attention. Heat stress not only causes apoptosis of spermatocytes but also has adverse effects on Sertoli cells, further damaging spermatogenesis. Lycium barbarum polysaccharide (LBP) is the main bioactive component of Lycium barbarum, which has a protective effect on male reproduction, but its mechanism is still unclear. In this study, our results proved that LBP blocked the inhibitory effect on the proliferation activity of Sertoli cells after heat stress, reversed the dedifferentiation of Sertoli cells induced by heat stress, and ameliorated the structural integrity of the blood-testis barrier. In addition, it increased the expression of the androgen receptor and activated Akt signaling pathway to resist heat-stress-induced injury of Sertoli cells.