Blood gas analysis in patients with varicocele
During laparoscopic varicocelectomy, peripheral venous blood and affected side venous blood of varicocele patients were extracted respectively for blood gas analysis to detect lactate concentration.
The establishment and assessment of the experimental rat varicocele model
Twenty adult male specific-pathogen free Sprague-Dawley rats (body weight, 250–300 g) were randomly assigned to a sham group (n=10) and a varicocele group (n=10). Rats were housed in a climate-controlled environment with free access to food and water under a 12-h day/night cycle. Experimental rat varicocele model was constructed according to our new clip technique . The establishment and assessment of the rat model is described in the previous articles published by our team. This study was approved by the ethics committee of the Third Affiliated Hospital of Southern Medical University and the animal work was approved by the Animal Care and Ethics Committee of Southern Medical University.
RNA isolation and qRT PCR analysis
Total RNA was extracted from the left testis of each animal using TRIzol reagent (Invitrogen, Carlsbad, USA) in accordance with the manufacturer’s instructions. In brief, tissues were homogenized and incubated in TRIzol for 10 min, and then RNA was separated using chloroform (0.2 mL/1 mL TRIzol). After centrifugation at 12,000 g for 15 min at 4°C, the aqueous phase of the sample was transferred to a fresh tube for RNA precipitation using isopropyl alcohol (0.5 mL/1 mL TRIzol). After repeating the incubation and centrifugation steps, the remaining pellet was washed with 75% ethanol and centrifuged at 7500 g for 5 min at 4°C. Finally, the air-dried pellet was re-dissolved in RNase-free water. The quantity and quality of the extracted RNA were measured with E-Spect (Malcon, Japan). In total, 200 ng of total RNA was reverse-transcribed with a PrimerScriptTM RT Kit (TaKaRa) for mRNA while quantitative real-time PCR for mRNA was performed in a 96-well plate using a SYBR Premix Ex Taq Real Time PCR Kit (TaKaRa). Amplification reactions were carried out in a final volume of 20 μL and were performed on an Mx3005P Stratagene under the following thermal cycling conditions: (denaturation at 95°C for 30 s [1×], followed by 40 cycles of denaturation [95°C, 30 s], annealing [60°C, 10 s] and extension [72°C, 15 s]). Rat Gapdh was used as an endogenous reference. The primers used for detecting Phgdh and Gapdh expression are shown in Table 1. The comparative cycle threshold method was performed for relative quantification. The sequences of the primers were as follows: PHGDH forward, 5'- GATGAAAGATGGCAAATGGGA -3′; PHGDH reverse, 5'- GCGGGGTATGGACAGTGATG -3'. GAPDH forward, 5′- GATGAAAGATGGCAAATGGGA -3′; GAPDH reverse5′- GCGGGGTATGGACAGTGATG -3′.
TM4 (a mouse testis Sertoli cell line) were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in DMEM with 10% FBS at 37°C in an incubator with an atmosphere of 5% CO2. For the experiments, TM4 were adherently cultured in 100-mm tissue culture dishes and reached 60~70% density before use.
Small interfering RNA (siRNA) and transient transfection
PHGDH siRNA was used to silence the PHGDH gene. A scrambled sequence siRNA (siNCtrl) was used as a negative control. The siRNA transfection was optimized using Lipofectamine2000-RNAimax (Invitrogen, Carlsbad, CA, USA), according to the manufacturer s instructions. Briefly, siRNA and lipofectamine were diluted separately in Opti-MEM (Gibco) and incubated at room temperature for 5 min. Then, the two solutions were gently mixed and incubated for 15 min. Finally, the mixture was added to plated cells, and after 48 hours, the cells were analyzed using the following assays.
Flow cytometry for cell apoptosis and cell cycle
PHGDH siRNA and a negative control siRNA were transfected as mentioned above.For cell apoptosis analysis, cells were prepared with the PE Annexin V Apoptosis Detection Kit I (BD Biosciences, New Jersey, USA) according to the manufacturer’s recommendations. For cell cycle analysis, cells were fixed and permeabilized by 75% ethanol, and were stained by PI/RNase Staining Buffer (BD Biosciences, New Jersey, USA) after incubation at −20°C overnight. The cell apoptosis ratio and cell cycle profile were detected by FAC Station (FV500,Beckman Coulter, Brea, USA), and the raw data were analyzed using FlowJo 10.0.7 software (FlowJo, Oregon, USA).
Western blot analysis
Total cells washed twice with cold PBS and lysed with RIPA (lysis buffer radioimmunoprecipitation assay) (Beyotime Institute of Biotechnology, China) and PMSF (protease inhibitors phenylmethanesulfonyl fluoride) (Beyotime Institute of Biotechnology, China). The protein concentration was determined by utilizing bicinchoninic acid (BCA) protein assay kit (P0010S, Beyotime Biotech, China). Equal amounts of protein (~10 μg) were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to 0.45 μm polyvinylidine difluoride filter (PVDF) membranes. The membranes were blocked with 5% BSA for 1h and then the membranes were incubated at 4°C overnight with primary antibodies. Next, the membranes were washed three times with TBST for at least 15minutes, and probed with HRP linked secondary antibodies for 1 h at room temperature. Protein bands were visualized employing the enhanced chemiluminescence detection kit (Thermo scientific, USA). The gray value of each protein band was analyzed by Image Lab software.
The primary antibodies were: PHGDH,PKM-2,PSPH and β-actin. All these antibodies were purchased from Abcam.
Glucose Consumption and Lactate Production
PHGDH siRNA and a negative control siRNA were transfected as mentioned above.After 48 hours, the media were collected for measurement of glucose and lactate concentrations as determined by glucose (GO) assay kit (Sigma) and lactate assay kit (Biovision). Glucose consumption and lactate production were normalized by cell numbers.
Measurement of LDH activities
PHGDH siRNA and a negative control siRNA were transfected as mentioned above. After 48 hours, the media were collected for measurement of LDH activities as determined by colorimetric assay kits in accordance with the manufacturer protocols.
Protein-protein interaction (PPI) network construction
Using the Search Tool for the Retrieval of Interacting Genes (STRING) database (http://www.string-db.org/), a PPI network related to PHGDH, PSPH, PSAT1 and PKM2 was established. The interactions procured included known interactions and predicted interactions.
Data were statistically analyzed using GraphPad Prism 8.0 software, and were presented as the mean±standard deviation (SD). An unpaired t-test was used to analyze the differences between the two groups. One-way ANOVA was used to analyze the intergroup differences among multiple groups. P < 0.05 was considered statistically significant.