Hyperoside protects mouse spermatocytes GC-2 cells from oxidative damage by activating the Keap1/Nrf2/HO-1 pathway / 南方医科大学学报

OBJECTIVE@#To study the protective effect of hyperoside (Hyp) against ydrogen peroxide (H2O2)- induced oxidative damage in mouse spermatocytes GC-2 cells and explore the role of the Keap1/Nrf2/HO-1 pathway in this protective mechanism.@*METHODS@#GC-2 cells were treated with 2.5 mmol/L azaacetylcysteine (NAC), 50, 100, and 200 μmol/L hyperoside, or the culture medium for 48 h before exposure to H2O2 (150 μmol/L) for 2 h. CCK-8 assay was used to detect the changes in cell viability, and cell apoptosis was analyzed using flow cytometry. Enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of superoxide dismutase (SOD), glutathione peroxidase (GSH-PX), catalase (CAT) activity and malondialdehyde (MDA) in the culture medium. Western blotting and RT-qPCR were used to detect the protein and mRNA expression levels of nuclear factor erythroid 2-related factor2 (Nrf2), Kelch-like ECH-associated protein 1 (Keap1), and heme oxygenase-1 (HO-1); the nuclear translocation of Nrf2 was detected using immunofluorescence assay.@*RESULTS@#Exposure to H2O2 significantly lowered the proliferation rate, reduced the activities of SOD, GSH and CAT, and obviously increased MDA content, cell apoptosis rate, and the expressions of Keap1 and Nrf2 mRNA and Keap1 protein in GC-2 cells (P < 0.05 or 0.01). Treatment of the cells prior to H2O2 exposure with either NAC or 200 μmol/L hyperoside significantly increased the cell proliferation rate, enhanced the activities of SOD, GSH-PX and CAT, and lowered MDA content and cell apoptosis rate (P < 0.05). Treatment with 200 μmol/L hyperoside significantly decreased the mRNA and protein expressions of Keap1 and increased the expressions of HO-1 mRNA and the protein expressions of Nrf2 and HO-1 (P < 0.05 or 0.01). Hyperoside also caused obvious nuclear translocation of Nrf2 in the cells (P < 0.05).@*CONCLUSION@#Hyperoside protects GC-2 cells against H2O2- induced oxidative damage possibly by activation of the Keap1/Nrf2/HO-1 signaling pathway.

Study on the mechanism of Wuzi-Yanzong-Wan-medicated serum interfering with the mitochondrial permeability transition pore in the GC-2 cell induced by atractyloside / 中国天然药物

Wuzi-Yanzong-Wan (WZYZW) is a classic prescription for male infertility. Our previous investigation has demonstrated that it can inhibit sperm apoptosis via affecting mitochondria, but the underlying mechanisms are unclear. The purpose of the present study was to explore the actions of WZYZW on mitochondrial permeability transition pore (mPTP) in mouse spermatocyte cell line (GC-2 cells) opened by atractyloside (ATR). At first, WZYZW-medicated serum was prepared from rats following oral administration of WZYZW for 7 days. GC-2 cells were divided into control group, model group, positive group, as well as 5%, 10%, 15% WZYZW-medicated serum group. Cyclosporine A (CsA) was used as a positive control. 50 μmol·L-1 ATR was added after drugs incubation. Cell viability was assessed using CCK-8. Apoptosis was detected using flow cytometry and TUNEL method. The opening of mPTP and mitochondrial membrane potential (MMP) were detected by Calcein AM and JC-1 fluorescent probe respectively. The mRNA and protein levels of voltage-dependent anion channel 1 (VDAC1), cyclophilin D (CypD), adenine nucleotide translocator (ANT), cytochrome C (Cyt C), caspase 3, 9 were detected by RT-PCR (real time quantity PCR) and Western blotting respectively. The results demonstrated that mPTP of GC-2 cells was opened after 24 hours of ATR treatment, resulting in decreased MMP and increased apoptosis. Pre-protection with WZYZ-medicated serum and CsA inhibited the opening of mPTP of GC-2 cells induced by ATR associated with increased MMP and decreased apoptosis. Moreover, the results of RT-qPCR and WB suggested that WZYZW-medicated serum could significantly reduce the mRNA and protein levels of VDAC1 and CypD, Caspase-3, 9 and CytC, as well as a increased ratio of Bcl/Bax. However, ANT was not significantly affected. Therefore, these findings indicated that WZYZW inhibited mitochondrial mediated apoptosis by attenuating the opening of mPTP in GC-2 cells. WZYZW-medicated serum inhibited the expressions of VDAC1 and CypD and increased the expression of Bcl-2, which affected the opening of mPTP and exerted protective and anti-apoptotic effects on GC-2 cell induced by ATR.

Simultaneous determination and analysis of amino acids and nucleosides in Dendrobium by UHPLC-QTRAP-MS/MS / 中国中药杂志

By using multivariate statistical analysis to evaluate essential quality, and provide scientific basis for their comprehensive utilization, we established an UHPLC-QTRAP-MS/MS method for the fast, precise, efficient determination of 21 kinds of amino acids and 10 kinds of nucleosides in different species of Dendrobium. The analysis was performed on a Waters XBridge Amide column(2.1 mm×100 mm,3.5 μm) with elution by mobile phase of 0.2% formic acid in water-0.2% formic acid in acetonitrile at a flow rate of 0.2 mL·min~(-1) with the column temperature at 30 ℃. The target compounds were analyzed by the positive ion multiple reaction monitoring(MRM) mode. The comprehensive evaluation of different species of Dendrobium was carried out by PCA and TOPSIS analysis. All 21 kinds of amino acids and 10 nucleosides showed good linearity among certain concentration range(r>0.999), the RSDs of the stability, precision, and repeatability tests were less than 3.0%. The recovery rate was in the range from 93.31% to 107.5%, and RSD was in the range of 1.1%-3.7%. The comprehensive evaluation index obtained with PCA showed that D. huoshanense was significantly higher than others regarding amino acids and D. officinale has higher nucleosides than other species. The biggest C_i difference of TOPSIS was 68.7%, and comprehensive evaluation showed that D. huoshanense produced the highest comprehensive quality. The method is precise, fast and efficient and can provide reliable basis for further researches and intrinsic quality control of Dendrobium.

Effect of Wuzi Yanzong Wan on Sperm Apoptosis by Intervening with Mitochondrial Permeability Transition Pore / 中国实验方剂学杂志

Objective:To study the effect of Wuzi Yanzong Wan on the expressions of voltage-dependent anion channel 1 （VDAC1）， adenine nucleotide transposase （ANT）， cyclophilin D （CypD） and other proteins， and analyze its mechanism in intervening with sperm mitochondrial permeability transition pore （mPTP） opening. Method:Forty rats were randomly divided into 4 groups， namely normal group， model group， positive group （Shengjing capsule， 1.6 g·kg-1·d-1）， and Wuzi Yanzong Wan group （4.0 g·kg-1·d-1）， with 10 rats in each group. Except for the normal group， tripterygium wilfordii glycosides （GTW， 30 mg·kg-1） was intragastrically administered for 8 weeks to establish the oligozoospermia model. After the 4th week， each group was given drugs through intragastric administration for 4 weeks， and fasted for 12 h after the last administration. These rats were anesthetized with 3% chloral hydrate， and their testis and epididymis tissues were collected. Western blot was used to determine the protein expressions of VDAC1，ANT，CypD，B-cell lymphoma/leukemia2 （Bcl-2）， Bcl-2 associated X protein （Bax）， Caspase-3， Caspase-9 in rat testis， Testicular tissue and its ultrastructure were observed under electron microscopy. The apoptosis in spermatogenic cells was detected by terminal-deoxynucleoitidyl transferase mediated nick end labeling （TUNEL）. Result:Western blot results showed that compared with the normal group， the expressions of VDAC1， CypD， Caspase-3， Caspase-9 and Bax/Bcl-2 in the model group were increased （P<0.01）. The expressions of VDAC1， CypD， Caspase-3， Caspase-9 and Bax/Bcl-2 were significantly decreased in the positive group and the Wuzi Yanzong Wan group compared with the model group （P<0.01）. There was no significant change in the expressions of ANT and Bcl-2 protein between the groups. Testicular ultrastructural evaluation showed different sizes and disordered arrangement of sperm mitochondria and a large number of swelling and vacuoles in the model group， while complete structure and neat arrangement of sperm mitochondria and much less swelling and vacuole in positive group and Wuzi Yanzong Wan group. TUNEL results showed that the apoptosis rate of spermatogenic cells in the model group was significantly higher than that of the normal group （P<0.01）， while the apoptosis rate in the positive drug and Wuzi Yanzong Wan group was significantly lower than that of the model group （P<0.01）. Conclusion:Wuzi Yanzong Wan may resist germ cell apoptosis by inhibiting the expressions of VDAC1， CypD and Bax， reducing the permeability of mPTP， and preventing the cascade activation reaction of the Caspase family of apoptosis proteins.