Objective: To investigate the mechanism of action of Wuzi Yanzong pill (WYP) in rats with oligoasthenozoospermia (OAZ) via metabolomics and to provide a possible basis for improving this WYP-based treatment. Methods: A rat model of OAZ was established by treating male Sprague–Dawley rats with glucosides from Tripterygium wilfordii Hook. F. Seventy-two rats were randomly divided into six groups: control, L-carnitine (positive control), model, and low-, medium-, and high-dose WYP groups. Rats in the experimental groups were treated with WYP for 4 weeks. At the end of the treatment period, sperm cell quality (density, motility, and viability) was assessed using a semen analysis system, mitochondrial membrane potential (MMP) was assessed using flow cytometry, and testicular injury was assessed using hematoxylin and eosin staining to validate the therapeutic effect of WYP in OAZ. Further, serum metabolomics-based analysis was performed using high-performance liquid chromatography-mass spectrometry to identify differential metabolic pathways and possible mechanisms of action of WYP in OAZ treatment. Results: A rat model of OAZ was considered successfully-established after comparing the quality of spermatozoa in the model group to that in the control group. WYP-M and WYP-H treatments significantly improved sperm cell density, motility, and viability compared with those in the model group (all P < .05). Compared with the model group, both WYP-M and WYP-H treatments increased MMP values (P = .006 and P = .021 respectively), while there was no significant difference in the L-carnitine group. L-carnitine and WYP administration reversed damage to the testes to varying degrees compared with that in the model group. Further, 44 differential metabolites and four metabolic pathways, especially autophagy pathway, related to OAZ were identified via metabolomics. Conclusions WYP improves sperm cell quality and MMP in OAZ primarily via autophagy regulation. These findings can be employed to improve the efficacy of WYP in humans.

Objective:To discuss the effect of Fuzheng Ruanjian Anticancer Formula on the malignant biological behaviors of the hepatocellular carcinoma HepG2 cells by requlating protein kinase B(Akt)/murine double minute 2(MDM2)/P53 signaling pathway.Methods:The HepG2 cells were treated with 0,0.05,0.10,0.20,0.40,0.80,1.60,3.20,and 6.40 g·mL-1 Fuzheng Ruanjian Anticancer Formula for 48 h.CCK-8 method was used to detect the survival rates of the HepG2 cells in various groups,and the concentrations of Fuzheng Ruanjian Anticancer Formula for the subsequent experiments were screened.The HepG2 cells were divided into control group,low dose of Fuzheng Ruanjian Anticancer Formula group(0.2 g·mL-1),medium dose of Fuzheng Ruanjian Anticancer Formula group(0.4 g·mL-1),high dose of Fuzheng Ruanjian Anticancer Formula group(0.8 g·mL-1),SC79 group(8 mg·L-1 SC79),and high dose of Fuzheng Ruanjian Anticancer Formula+SC79 group(0.8 g·mL-1 Fuzheng Ruijian Anticancer Formula+8 mg·L-1 SC79).CCK-8 method was used to detect the proliferation activities of the HepG2 cells in various groups;clone formation assay was used to detect the clone formation rates of the HepG2 cells in various groups;flow cytometry was used to detect the apoptotic rates of the HepG2 cells in various groups;Transwell chamber assay was used to detect the numbers of migration and invasion HepG2 cells in various groups;Western blotting method was used to detect the expression levels of proliferating cell nuclear antigen(PCNA),cysteine aspartate specific proteinase(Caspase-3),matrix metalloproteinase(MMP)-2,MMP-9,phosphorylated Akt(p-Akt),phosphorylated MDM2(p-MDM2),and P53 proteins in the HepG2 cells in various groups.Results:As the increasing of concentrations of Fuzheng Ruanjian Anticancer Formula(0,0.05,0.10,0.20,0.40,0.80,1.60,3.20,and 6.40 g·mL-1),the surival rates of the HepG2 cells were gradually decreased(P<0.05),and 0.2,0.4,and 0.8 g·mL-1 Fuzheng Ruanjian Anticancer Formula were selected for the subsequent experiments.The CCK-8 assay results showed that compared with control group,the proliferation activities of the HepG2 cells in low,medium,and high doses of Fuzheng Ruanjian Anticancer Formula groups were significantly decreased(P<0.05),in a dose-dependent manner,while the proliferation activity of the cells in SC79 group was significantly increased(P<0.05).Compared with high dose of Fuzheng Ruanjian Anticancer Formula group,the proliferation activity of the HepG2 cells in high dose of Fuzheng Ruanjian Anticancer Formula+SC79 group was significantly increased(P<0.05).The clone formation assay results showed that compared with control group,the clone formation rates of the HepG2 cells in low,medium,and high doses of Fuzheng Ruanjian Anticancer Formula groups were significantly decreased(P<0.05)in a dose-dependent manner,while the clone formation rate of the cells in SC79 group was significantly increased(P<0.05);compared with high dose of Fuzheng Ruanjian Anticancer Formula group,the clone formation rate of the cells in high dose of Fuzheng Ruanjian Anticancer Formula+SC79 group was significantly increased(P<0.05).The flow cytometry results showed that compared with control group,the apoptotic rates of the HepG2 cells in low,medium,and high doses of Fuzheng Ruijian Anticancer Formula groups were significantly increased(P<0.05)in a dose-dependent manner,while the apoptotic rate of the cells in SC79 group was significantly decreased(P<0.05);compared with high dose of Fuzheng Ruanjian Anticancer Formula group,the apoptotic rate of the cells in high dose of Fuzheng Ruanjian Anticancer Formula+SC79 group was significantly decreased(P<0.05).The Transwell chamber assay results showed that compared with control group,the numbers of migration and invasion HepG2 cells in low,medium,and high doses of Fuzheng Ruanjian Anticancer Formula groups were significantly decreased(P<0.05)in a dose-dependent manner,while the numbers of migration and invasion cells in SC79 group were significantly increased(P<0.05);compared with high dose of Fuzheng Ruanjian Anticancer Formula group,the numbers of migration and invasion HepG2 cells in high dose of Fuzheng Ruanjian Anticancer Formula+SC79 group were significantly increased(P<0.05).The Western blotting results showed that compared with control group,the expression levels of PCNA,MMP-2,MMP-9,p-Akt,and p-MDM2 proteins in the cells in low,medium,and high doses of Fuzheng Ruanjian Anticancer Formula groups were significantly decreased(P<0.05)in a dose-dependent manner,while the expression levels of Caspase-3 and P53 proteins were significantly increased(P<0.05)in a dose-dependent manner,while the expression levels of PCNA,MMP-2,MMP-9,p-Akt,and p-MDM2 proteins in the cells in SC79 group were significantly increased(P<0.05),and the expression levels of Caspase-3 and P53 proteins were significantly decreased(P<0.05);compared with high dose of Fuzheng Ruanjian Anticancer Formula group,the expression levels of PCNA,MMP-2,MMP-9,p-Akt,and p-MDM2 proteins in the cells in high dose of Fuzheng Ruanjian Anticancer Formula+SC79 group were significantly increased(P<0.05),while the expression levels of Caspase-3 and P53 proteins were significantly decreased(P<0.05).Conclusion:Fuzheng Ruanjian Anticancer Formula may inhibit the proliferation,migration,and invasion of the HepG2 cells and promote the apoptosis,and its mechanism may be related to suppressing the Akt/MDM2 signaling pathway and upregulating the P53 proteim expression.