Drugs and reagents
S. miltiorrhiza radix (lot 1805031) was obtained from Beijing Jin Chongguang Pharmaceutical Co., Ltd. (Beijing, China), Tanshinone IIA (Tan IIA) ( ＞98% purity; lot 20170908), salvianic acid A sodium (SAS) (＞98% purity; lot 171001) was obtained from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Acetazolamide (ACZ) (＞98% purity; lot B27044) was purchased from Beijing BioDee Biotechnology Co., Ltd. (Beijing, China). LY294002 (＞99.5% purity, CAS No.154447-36-6) was purchased from MedChemExpress LLC(USA, New Jersey). Kits of glutathione peroxidase (GSH-Px, Cat. No. A005), total superoxide dismutase (T-SOD, Cat. No. A001-1), Catalase (CAT), and malondialdehyde (MDA, Cat. No. A003-1) were purchased from Nianjing Jiancheng Bioengineering Institute (Nanjing, China); Hypoxia Inducible Factor 1 α (HIF-1α) was purchased from Quanzhou Konodi Biotechnology Co., Ltd. RPMI-1640 and fetal bovine serum (FBS) are Hyclone products obtained from Thermo Fisher Scientific (Waltham, MA, USA). Dichlorofluorescein diacetate and a Cell Counting Kit‑8 (CCK‑8) were obtained from Sigma Millipore (Darmstadt, Germany). Apoptosis detection kit and fluo‑3‑acetoxymethyl ester were purchased from Biosea Biotechnology (Beijing, China). The reactive oxygen species (ROS) fluorescent probe, dihydroergotamine, was purchased from Vigorous Biotechnology (Beijing, China). TransScript™ First-Step quantitative reverse transcriptase polymerase chain reaction SuperMix and SuperMix were purchased from Promega Corporation (Fitchburg, WI, USA). Applied Biosystems Fast [email protected] Green Master Mix was purchased from Thermo Fisher Scientific. The tissue protein extraction kit was obtained from Cowin Biotech Company Ltd. (Beijing, China). bicinchoninic acid (BCA) was purchased from Applygen Technologies Inc (Beijing, China). Polyvinylidene difluoride membrane and the ECL® kit were purchased from Amersham (now GE Healthcare, Arlington Heights, IL, USA). Protein markers were purchased from Genstar (Beijing, China). Ngb Polyclonal Antibody (PA5-76950) were purchased from Invitrogen Corporation (Carlsbad, USA). Akt Polyclonal Antibody(YT0175), Akt (phospho Ser473) Polyclonal Antibody(YP0006) were purchased from ImmunoWay Biotechnology Company(Texas, USA) Other analytical reagents were purchased from Promega (Beijing) Biotech Co., Ltd. (Beijing, China), low pressure and low oxygen animal test cabin borrowed from the Academy of military medicine (model: flydwc50-1b, Guizhou Fenglei Aviation Ordnance Co., Ltd., China)
Preparation of Decoctions.
Fifty grams of S. miltiorrhiza were extracted using deionized water and ethanol (1:1, 500 mL) for 1 h during microboiling under reﬂux. The extracts were filtered through three layers of gauze, and the drug extraction was then repeated. The filtrates were combined and were then concentrated to 250 mL at 60–70°C under reduced pressure.
For the in vivo experiments, samples were shaken, calibrated, and stored at 4℃. Based on a preliminary test, ACZ was prepared with normal saline according to 60 mg/mL. Tan ⅡA was prepared with soybean oil for injection at a concentration of 6 mg/mL. SAS was prepared with soybean oil for injection at 12 mg/mL.
For the in vitro experiments, SE was centrifuged at 1200 rpm for 3 min and filtered with 0.2 μM filter membrane to prepare 1 g/mL in Phosphate buffer solution (PBS), SAS, and Tan IIA dissolved in Dimethyl sulfoxide (DMSO). Through the CCK-8 test, we chose the dose of SE (1 mg/mL), Tan ⅡA (40 μM), and SAS (100 μM), which has no toxic effect on cell viability according to the follow-up test.
Animals and treatments
Seven-week-old male BALB/c mice weighing 20–25 g were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd. and fed at the specific pathogen free (SPF) level animal center of the Academy of Military Medical Sciences (Beijing, China). The mice were kept in an SPF animal facility at a constant temperature of 23°C and relative humidity of 40%–60%. The protocol was approved by the Committee on the Ethics of Animal Experiments of the animal center of the Academy of Military Medical Sciences. The animal experiments were conducted in accordance with the Guiding Principles in the Use of Animals in Toxicology.
Group assignment and drug administration
Mice were randomly divided into six groups (n=8 per group): vehicle, ACZ, SE, SAS, and Tan IIA groups. Animals were treated with ACZ, SE, SAS, and Tan IIA for 10 days by intraperitoneal injection or gastric lavage as shown in Table 1. Continuous administration lasted for 10 days. On the 4th day, 30 min after daily administration, the rats were exposed to hypobaric hypoxia, which increased at a rate of 10 m/s and was maintained at 3000 m for 10 min; and then increased to 4500 m and stayed for 10 min; final increased to 6000 m, after 16 h of hypoxia exposure, it decreased to sea level at a rate of 15 m/s. Mice in the control group were fed in normoxic environment. After the last hypoxia exposure for 15 h, the mice were dissected. Group assignment and drug administration as shown in Table 1.
Daily changes in body weight and clinical signs. For macroscopic damage, brain were fixed in 10% buffered formalin. A single pathologist, who was blinded to the treatment, duration, and genotype of the sample, examined and scored brain representative from three mice per group. Hematoxylin and eosin (H&E)- Nissl-stained sections were assessed according to a standard staining protocol.
Determination of the levels of MDA / HIF-1α and activities of T-SOD, GSH-Px, and CAT
Mice were sacrificed by cervical dislocation. The blood of each mouse was obtained from the aortaventralis. The brain were immediately removed and washed with ice-cold normal saline. The left brain were weighed and stored at -80°C, and then homogenized with a phosphate buffer (50 mmol/L, pH 7.0) containing 0.1 mmol/L EDTA before use. The brain homogenate was divided into two parts. One part was centrifuged at 2054 g for 10 min at 4°C, and the supernatant was used for MDA, GSH-Px, T-SOD, and CAT tests. Another part was used for direct determination of HIF-1α activity. The blood and brain homogenate were centrifuged at 2054 g for 10 min at 4°C to obtain serum and brain supernatant for MDA, GSH-Px, T-SOD, CAT, and HIF-1α tests. All parameters were determined using the respective kits according to the manufacturer’s specifications.
Cell culture and treatment
PC12 human neuroblastoma cells were grown in RPMI-1640 supplemented with 15% FBS. Cells were maintained at 37°C in an incubator with a saturated humid atmosphere containing 95% air and 5% CO2. Cells were passaged once every three days. All experiments were conducted on cells between passages 10-20 after cells were purchased from National Infrastructure of Cell Resource (Beijing, China).
Cell viability analysis
Cytotoxicity was assayed in PC12 cells grown in 96‑well plates. Cells (1.5x105 cells/ml; 0.1 ml per well) were seeded into plates and allowed to grow overnight before replacement of the medium with serum‑free medium supplemented with CoCl2 1mM co‑treated with drug for 24h. Subsequently, 10 μl CCK‑8 was added into each well and incubated for 1‑4 h. The absorbance was read at 450 nm with a PerkinElmer Victor X Microplate Reader (PerkinElmer, Inc., Waltham, MA, USA). Reductions in optical density (OD) due to drug treatment were used to assess cell viability and normalized against control incubated in medium (100% viability).
Measurement of the mitochondrial membrane potential (MMP)
MMP was measured using flow cytometry, and the mitochondrial‑specific cationic dye, JC‑1. PC12 cells (1.5x105 cells/ml) were plated in 12‑well plates CoCl2 1mM co‑treated with drug for 24h. Cells were harvested, washed twice with PBS, and incubated with 0.5 mL JC‑1 (25 μM) for 20 min at 37°C. MMP was assayed, and green (JC‑1 monomer) and red (JC‑1 aggregate) fluorescence were monitored at emission wavelengths of 525 and 595 nm, respectively. Changes in the ratio between measurements were indicative of changes in the MMP.
Measurements of intracellular ROS and Ca2+
PC12 cells (1.5x105 cells/ml) were plated in 12‑well plates with CoCl2 1mM co‑treated with drug for 24h, and Intracellular ROS and cytosolic Ca2+ were measured using the fluorescent probes DCFH‑DA and Fluo‑3‑AM, respectively, and a fluorescence‑activated cell sorter. DCFH‑DA is converted into a fluorescent compound in the presence of ROS. Fluo‑3‑AM was added to treated cells to measure Ca2+. After treatment with the indicated drugs, cells were incubated with DCFH‑DA (10 μM) for 20 min at 37°C in the dark (for the ROS assay) or Fluo‑3/AM (5 μmol/l) for 30 min at 37°C (the Ca2+ assay), and the cells were then harvested and suspended in 500 μl HBSS. Intracellular ROS and Ca2+ were measured using a flow cytometer (excitation wavelength, 488 nm; emission wavelength, 535 nm).
Apoptosis was also measured using annexin V‑FITC and PI. PC12 cells were plated (1.5x105 cells/ml; 0.1 ml) in 12‑well plates with CoCl2 1mM co‑treated with drug for 24h. Cells were harvested, washed twice with ice‑cold PBS, and then suspended in 200 μl ice‑cold binding buffer. Subsequently, 10 μl HRP FITC‑labeled annexin V and 5 μL PI were added to the cells. The cell suspension was gently mixed, and incubated for 15 min at room temperature in the dark. Apoptosis was monitored using flow cytometry (488 nm excitation wavelength), and the fluorescence intensity was measured at 530 nm (emission wavelength). Annexin V+/PI‑ was used to document early apoptosis, whereas AnnexinV+/PI+ was used to assess the late apoptotic stages or necrotic cells.
Quantitative real-time PCR
Total RNA was extracted from brain tissue and PC12 cells using Invitrogen® TRIzol reagent according to the manufacturer's instruction (Thermo Fisher Scientific, Inc.). PC12 cells were maintained in 6-well plates until 80% conﬂuence and then treated with Tan IIA、SAS and SE in serum-free medium for various periods of time to assess the specific induction of Ngb expression. CoCl2 (1 mM) and dimethyl sulfoxide (DMSO) was used as the control. The quality of the RNA was confirmed by an A260/A280 ratio of＞ 1.8 and an RNA integrity number ≥6.5.Complementary DNA (cDNA) was synthesized using a Transcriptor™ first-strand cDNA synthesis kit at 42°C for 30 min followed by 70°C for 5 min. qRT-PCR reactions were performed on an ABI StepOne Plus™ Real Time PCR instrument using SYBR Green PCR Master Mix. The amplification reactions were performed as follows: 20 seconds at 95°C, and 40 cycles at 95°C for 3 seconds, and 60°C for 30 seconds. The primers used in the current study are listed in Table 2. The quantity of each transcript was calculated as described in the instrument manual and was normalized to the amount of the housekeeping gene β-actin.
Preparation of total protein and Western blotting
Total protein was extracted from brain tissue and PC12 cells according to the instruction. PC12 cells (1.5x105 cells/ml) were plated in 6‑well plates with CoCl2 1mM for 24h, and then treated with SE(1g/ml), SAS(50mM), Tan IIA(40mM) and LY294002(10mM) co‑treated with SE(1g/ml), SAS(50mM), Tan IIA(40mM) for 24h. The protein expression of Ngb, Hif-1α, Akt and P-Akt were determined by Western blotting. Protein of brain and PC12 cells were extracted with ice‐cold lysis buffer and centrifuged at 12,000 × g for 10 min., and the resultant supernatant assayed using BCA protein assay kit standardized to BSA. Equal amounts of total protein (40 μg) were loaded, separated by 12% SDS‐PAGE, and transferred to polyvinylidene fluoride (PVDF) membrane. The membranes were blocked in 5% TBST fat free milk (blocking buffer) for 2 hrs, briefly washed, and then incubated overnight at 4°C with specific primary antibodies against human Ngb (PA5-76950; dilution 1:500), Hif-1α (dilution 1:500), Akt (YT0175; dilution 1:1000), P-Akt (YP0006; dilution 1:1000). The samples were subsequently incubated with monoclonal IgG (1:2,000) secondary antibody for 2 hours, and visualized on film using a Santa Cruz ECL detection system. β-actin served as a loading control. The blank control group of mice and the DMSO solvent control group of PC12 cells served as the negative controls respectively. Densitometric analyses were performed to semiquantify protein expression.